Method for producing reduced coenzyme Q10 by fermentation of recombinant strains

A technology of recombinant strains and coenzymes, applied in the field of molecular biology, to achieve the effects of low raw material cost, large-scale production, and simple separation process

Inactive Publication Date: 2012-09-19
苏州海吉亚生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] As mentioned above, there are currently no examples of high-yielding reduced coenzyme Q10 strains obtained through genetic recombination, let alone the use of recombined reducing strains for industrial-scale fermentation of reduced coenzyme Q10.

Method used

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  • Method for producing reduced coenzyme Q10 by fermentation of recombinant strains
  • Method for producing reduced coenzyme Q10 by fermentation of recombinant strains

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] 1. Gene recombination of Rhodotorula glutinis (IFO 1125)

[0029] The operation steps are as follows:

[0030] (1) Cloning Rhodotorula glutinis (IFO 1125) 1-deoxy-D-xylulose-5-phosphate synthase (dxs11) gene and decaprenyl diphosphate synthase (dps44) gene.

[0031] (2) Design two pairs of primers to add restriction sites BamHI and XhoI: DXS up: 5'-TTTGGATCCTTGACCGGAATGCCACAG-3', DXSdown: 5'-TTTCTCGAGTCAGCCGGCGAAACCGAC-3', DPSup: 5'-TTTGGATCCTTGCCGCGCAAGGCGTCA-3' DPSdown: 5' -TTTCTCGAGTCAGTTGAGACGCTCGAT-3'.

[0032] (3) Genomic DNA of Rhodotorula glutinis (IFO 1125) was extracted, and DXS and DPS genes were amplified by PCR.

[0033](4) After adding A to the end of the DXS and DPS genes, connect them to the T vector, transform into DH5a, pick the clones and shake the bacteria to extract the plasmids to obtain the correct recombinant plasmids, and then use BamHI and XhoI to digest and recover the above two gene fragments.

[0034] (5) Digest the prokaryotic expression...

Embodiment 2

[0056] 1. The gene recombination operation steps of Agrobacterium radiobacter (ATCC4718) are as follows:

[0057] (1) Clone Agrobacterium radiobacter (ATCC4718) 1-deoxy-D-xylulose-5-phosphate synthase (dxs) gene and 1-Deoxy-D-xylulose-5-phosphate reductoisomerase (dxr) gene.

[0058] (2) Design two pairs of primers to add restriction sites BamHI and XhoI: DXSup: 5'-TTTGGATCCTTGACCGGAATGCCACAG-3', DXSdown: 5'-TTTCTCGAGTCAGCCGGCGAAACCGAC-3', DXRup: 5'-TTTGGATCCATGACGAATGCCAGTGAA-3' DXRdown: 5'- TTTCTCGAGTCACGCGGCCTTTTCTTT-3'.

[0059] (3) Genomic DNA of Agrobacterium radiobacter (ATCC4718) was extracted, and DXS and DXR genes were amplified by PCR.

[0060] (4) Add A to the ends of DXS and DXR genes and connect them with T vectors, transform DH5a, pick clones and shake bacteria to extract plasmids to obtain correct recombinant plasmids, and then use BamHI and XhoI to digest and recover the above two gene fragments.

[0061] (5) Digest the prokaryotic expression vectors PET-24-...

Embodiment 3

[0082] 1. Gene recombination of Rhodotorula glutinis (IFO 1125)

[0083] The operation steps are as follows:

[0084](1) Clone Rhodotorula glutinis (IFO 1125) 1-deoxy-D-xylulose-5-phosphate synthase (dxs) gene, decaprenyl diphosphate synthase (dps) gene and 1-Deoxy-D-xylulose-5-phosphate reductoisomerase (dxr) gene.

[0085] (2) Design three pairs of primers to add restriction sites BamHI and XhoI: DXS up: 5'-TTTGGATCCTTGACCGGAATGCCACAG-3', DXSdown: 5'-TTTCTCGAGTCAGCCGGCGAAACCGAC-3', DPSup: 5'-TTTGGATCCTTGCCGCGCAAGGCGTCA-3' DPSdown: 5' -TTTCTCGAGTCAGTTGAGACGCTCGAT-3', DXRup: 5'-TTTGGATCCATGACGAATGCCAGTGAA-3'DXRdown: 5'-TTTCTCGAGTCACGCGGCCTTTTCTTT-3'.

[0086] (3) Genomic DNA of Rhodotorula glutinis (IFO 1125) was extracted, and DXS, DPS and DXR genes were amplified by PCR.

[0087] (4) Add A to the end of DXS, DPS and DXR genes and connect them with T vector, transform DH5a, pick clones and extract plasmids to obtain correct recombinant plasmids, and then use BamHI and XhoI...

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Abstract

The invention discloses a method for producing reduced coenzyme Q10 by fermentation of recombinant strains, belonging to the technical field of molecular biology. The method comprises the steps of transferring genes for producing the reduced coenzyme Q10 into strains with reduced coenzyme Q10 ratio of more than 70%; using mutagenesis technology to screen strains with reduced coenzyme Q10 ratio of more than 90%; using the strains with the reduced coenzyme Q10 ratio of more than 90% as cultivated strains to produce the reduced coenzyme Q10. The method further improves production capacity of the gene recombinant strains by using high-yield strains built by gene engineering and mutagenesis technology, so that the method can satisfy requirement of the commercial production.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method for fermenting and producing reduced coenzyme Q10 by recombinant strains. Background technique [0002] Reductive coenzyme Q10 is an effective antioxidant with a wide range of uses. In developed countries, it has been widely used in food additives, daily chemical products, health products, cosmetics and other fields in addition to medicines. It has many medicinal effects, and it has been clinically proven to eliminate fatigue, enhance physical strength, anti-aging, immune regulation, anti-tumor effect, relieve Parkinson's disease, treat and relieve nervous headaches, and reduce the side effects of doxorubicin or statins Wait for the effect. [0003] Reductive coenzyme Q10 and oxidative coenzyme Q10 are the constituent factors of the mitochondrial electron conduction system in human cells, which play the role of transporting electrons in the oxidati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/66C12N1/21C12R1/01
Inventor 糜军党珍李明
Owner 苏州海吉亚生物科技有限公司
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