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Genetically engineered bacteria and application thereof in production of coenzyme Q10

A genetically engineered bacteria and gene technology, applied to genetically engineered bacteria and its application in the production of coenzyme Q10, can solve problems such as the need to improve coenzyme Q10, and achieve the effect of increasing yield

Active Publication Date: 2016-03-30
SHANGYU NHU BIOCHEM IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, the ability of the genetically engineered bacteria obtained through metabolic engineering to synthesize coenzyme Q10 needs to be improved.

Method used

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  • Genetically engineered bacteria and application thereof in production of coenzyme Q10
  • Genetically engineered bacteria and application thereof in production of coenzyme Q10
  • Genetically engineered bacteria and application thereof in production of coenzyme Q10

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] The construction of embodiment 1 recombinant plasmid

[0053] 1 Design primers

[0054] Primer sequences for amplifying three target genes were designed with Primer5 primer design software, wherein,

[0055] The primers used to amplify nornaquinone methyltransferase gene (UbiE) are:

[0056] Upstream primer: 5'- TCTAGA GCATCAACGGAGGTTCAGGGTGGTGAATGAGCGACGAAACTTCCAAC-3';

[0057] Downstream primer: 5'-CCC AAGCTT CGAGCTCCGGCCGCTACTAGTTGCGAACCAGCCGCCAGA-3';

[0058] Wherein, the upstream primer has an XbaI restriction site (underlined part), and the downstream primer has a HindIII restriction site (underlined part);

[0059] The primers used to amplify 3-demethylubiquinone-93-methyltransferase gene (UbiG) are:

[0060] Upstream primer: 5'-CC AAGCTT CATCAACGGAGGAGGAGTTTGCAATGGAATCGT-3';

[0061] Downstream primer: 5'-GG ACTAGT TCAGCTGCGCCGCACGCTCGCGGTAACGT-3';

[0062] Wherein, the upstream primer is added with a HindIII restriction site (underlined part), and ...

Embodiment 2

[0104] Embodiment 2 constructs genetically engineered bacteria

[0105] 1 Recombinant plasmid transformed into Escherichia coli S17-1

[0106] Take out Escherichia coli S17-1 competent 2 tubes, add recombinant plasmid pBBR1MCS-2-UbiE-UbiG-UbiF after 10 minutes of ice bath, ice bath for 20 minutes, heat shock for 90 seconds, ice bath for 5 minutes, add 600 μl LB liquid medium, After incubating at 37°C for 45 minutes, centrifuge at 5000 r / min for 5 minutes, discard 300 μl of the supernatant, and spread the remaining liquid onto the Kanna plate.

[0107] 2 joint transfer

[0108] 1) Inoculate Rhodobacter sphaeroides CGMCC No.5997 into a test tube containing 10ml of NHU-liquid medium, and culture at 30°C and 200r / min for 36h. The formula of NHU-liquid medium is (100ml): yeast extract 0.8g, FeSO 4 0.01g, K 2 HPO 4 0.13g, CoCl 2 0.003g, NaCl0.2g, MnSO 4 0.0001g, MgSO 4 0.025g, glucose 0.3g, vitamin B 10.1μg, vitamin K 0.1μg, vitamin A 0.15μg, pH adjusted to 7.2.

[0109] 2)...

Embodiment 3

[0135] Embodiment 3 Utilizes genetically engineered bacteria to prepare coenzyme Q10

[0136] Utilize the genetically engineered bacteria NHU-EFG strain obtained in Example 2 to carry out fermentation, and use the original unmodified Rhodobacter sphaeroides CGMCCNo. Rhodobacter sphaeroids NHU-F, Rhodobacter sphaeroids NHU-G with only UbiG gene, and Rhodobacter sphaeroids NHU-EG with only UbiE and UbiG genes were used as controls to compare the production of coenzyme Q10 in the four strains.

[0137] The specific fermentation method is as follows:

[0138]1. Primary seed culture: Pick a single clone (positive clone) of the NHU-EFG strain and inoculate it in a 250ml shake flask containing 50ml seed culture solution, and cultivate it at 30°C and 200r / mn for 23h to obtain the first-grade seed solution;

[0139] The formula of seed culture solution is (100ml): (NH 4 ) 2 SO 4 0.25g, corn steep liquor 0.05g, yeast extract 0.14g, NaCl 0.2g, glucose 0.3g, K 2 HPO 4 0.05g, KH 2 P...

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Abstract

The present invention discloses genetically engineered bacteria and application thereof in production of coenzyme Q10. The genetically engineered bacteria comprises coenzyme Q10 producing bacteria and desired genes transferred into the coenzyme Q10 producing bacteria, and the desired genes are a demethylnaphthoquinone methyltransferase gene, a 2-octo isoprene-3-methyl-6-methoxy-1,4-hydroquinone hydroxylase gene and a 3-demethyl ubiquinone-93-methyl transferase gene. Rate-limiting enzyme genes UbiE, UbiG and UbiF in a coenzyme Q10 synthetic route are transferred into rhodobacter sphaeroides, and by in-series over-expression of the three rate-limiting enzyme genes UbiE, UbiG and UbiF, coenzyme Q10 synthesizing capability of the rate-limiting enzyme genes can be strengthened; and compared with the rhodobacter sphaeroides with no desired gene being transformed, by use of the genetically engineered bacteria for coenzyme Q10 producing, the yield of the coenzyme Q10 is increased by more than 20%.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a genetically engineered bacterium and its application in the production of coenzyme Q10. Background technique [0002] Coenzyme Q is a fat-soluble quinone compound that exists widely in organisms. Coenzyme Q from different sources has different numbers of isopentenyl units in its side chain. Humans and mammals have 10 isopentenyl units, so it is called coenzyme Q10. Its structural formula is as follows Shown in formula (I): [0003] [0004] Coenzyme Q10 is an important hydrogen transporter in the respiratory chain of biological cells and a good biochemical drug. In recent years, it has been widely used in the treatment of various diseases such as heart disease, diabetes, cancer, acute and chronic hepatitis, and Parkinson's disease. In addition, it also has significant effects in treating scurvy, duodenal ulcer, necrotizing periodontitis, and promoting pancreatic function and se...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P7/66C12R1/01
Inventor 陈召峰胡伟江于凯朱永强于洪巍陆文强杨梢烽吕家樑
Owner SHANGYU NHU BIOCHEM IND
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