Method for constructing high-yield coenzyme Q10 engineering bacteria and application of method

A technology of engineering bacteria and coenzymes, which is applied in the field of genetic engineering, can solve the problems of low yield and achieve the effects of increasing potency, promoting synthesis, and increasing yield

Inactive Publication Date: 2019-09-13
兰州天禾生物催化技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a method for constructing high-yield coenzyme

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1, the method for constructing high-yielding coenzyme Q10 engineering bacteria is as follows:

[0042](1) Select Rhodobacter sphaeroides containing the gene encoding isopentenyl pyrophosphate isomerase IDI as the parent strain, and use the genomic DNA extraction kit to extract the genomic DNA of Rhodobacter sphaeroides.

[0043] (2) The upstream primer IDIF and the downstream primer IDIR were designed by Primer5 primer design software, the upstream primer added the restriction site NcoI, and the downstream primer added the restriction site BamHI.

[0044] (3) Using the extracted genomic DNA as a template, the target gene was obtained by PCR amplification. A standard reaction system was used: 25 μL of GC buffer, 16 μL of water, 4 μL of dNTP mixture, 1.5 μL (10uM) of upstream primer IDIF, 1.5 μL (10uM) of downstream primer IDIR, 1.5 μL of extracted genomic DNA, and 0.5 μL of PrimeSTAR enzyme. The amplification program was: 30 cycles, each cycle including denat...

Embodiment 2

[0051] Embodiment 2, the method for constructing high-yielding coenzyme Q10 engineering bacteria is as follows:

[0052] (1) Select Rhodopseudomonas capsulatum containing the gene encoding isopentenyl pyrophosphate isomerase IDI as the parent strain, and use the genomic DNA extraction kit to extract the genomic DNA of Rhodopseudomonas capsulatum.

[0053] (2) The upstream primer IDIF and the downstream primer IDIR were designed by Primer5 primer design software, the upstream primer added the restriction site NcoI, and the downstream primer added the restriction site BamHI.

[0054] (3) Using the extracted genomic DNA as a template, the target gene was obtained by PCR amplification. A standard reaction system was used: 25 μL of GC buffer, 16 μL of water, 4 μL of dNTP mixture, 1.5 μL (10uM) of upstream primer IDIF, 1.5 μL (10uM) of downstream primer IDIR, 1.5 μL of extracted genomic DNA, and 0.5 μL of PrimeSTAR enzyme. The amplification program was: 30 cycles, each cycle includ...

Embodiment 3

[0061] Embodiment 3, the method for constructing high-yielding coenzyme Q10 engineering bacteria is as follows:

[0062] (1) Agrobacterium tumefaciens containing the gene encoding isopentenyl pyrophosphate isomerase IDI was selected as the parent strain, and genomic DNA of Agrobacterium tumefaciens was extracted using a genomic DNA extraction kit.

[0063] (2) The upstream primer IDIF and the downstream primer IDIR were designed by Primer5 primer design software, the upstream primer added the restriction site NcoI, and the downstream primer added the restriction site BamHI.

[0064] (3) Using the extracted genomic DNA as a template, the target gene was obtained by PCR amplification. A standard reaction system was used: 25 μL of GC buffer, 16 μL of water, 4 μL of dNTP mixture, 1.5 μL (10uM) of upstream primer IDIF, 1.5 μL (10uM) of downstream primer IDIR, 1.5 μL of extracted genomic DNA, and 0.5 μL of PrimeSTAR enzyme. The amplification program was: 30 cycles, each cycle inclu...

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Abstract

The invention discloses a method for constructing high-yield coenzyme Q10 engineering bacteria and an application of the method, belongs to the technical field of genetic engineering, and solves the problem of low yield by an existing coenzyme Q10 production method. The method disclosed by the invention comprises the following steps of performing extraction to obtain genome DNA from a gene parentstrain containing coding isopentenyl diphosphate isomerase IDI; amplifying out a homologous gene of the isopentenyl diphosphate isomerase IDI through a polymerase chain reaction; enabling the amplified homologous gene to be connected with a plasmid vector, and constructing a recombinant carrier; and guiding the recombinant carrier into a host cell so as to obtain target engineering bacteria. The invention provides an application of the method for constructing the high-yield coenzyme Q10 engineering bacteria to production of a coenzyme Q10. According to the method disclosed by the invention, through constructing the isopentenyl diphosphate isomerase IDI overexpressed by engineering bacteria, precursor substances synthesized by the coenzyme Q10 are increased, the synthesis capacity of the coenzyme Q10 can be greatly increased, the yield of the coenzyme Q10 is notably increased, and the method can be used for large-scale industrial production of the coenzyme Q10.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for constructing high-yielding coenzyme Q10 engineering bacteria and its application. Background technique [0002] Coenzyme Q10 (CoQ10) is also known as ubiquinone and decenoquinone, and its chemical name is 2,3-dimethoxy-5-methyl-6-decenylbenzoquinone. The biological activity of coenzyme Q10 comes from the oxidation-reduction properties of its quinone ring and the physicochemical properties of its side chain. It is a natural antioxidant and cell metabolism activator produced by cells itself. It has anti-oxidation, eliminates free radicals, and improves the body's immunity. , anti-aging and other functions, it is widely used clinically in the treatment of various diseases such as heart disease, cancer, diabetes, acute and chronic hepatitis, Parkinson's disease, etc., and it also has many applications in food, cosmetics and anti-aging health care ...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/81C12N15/74C12P7/66C12R1/19C12R1/01C12R1/74
CPCC12N15/70C12N15/81C12N15/74C12P7/66
Inventor 高维东谢小冬弥超秦云刘恭骆小军张调调刘慧琴
Owner 兰州天禾生物催化技术有限公司
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