Coenzyme-Q10-production engineered bacteria construction method, engineered bacteria, and application thereof

A construction method and technology for engineering bacteria are applied in the construction field of producing coenzyme Q10 engineering bacteria, which can solve the problem of high cost and achieve the effects of high synthesis capacity, good effect of large-scale production and application, and low pollution.

Active Publication Date: 2014-01-15
ZHEJIANG NHU CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although it is ideal to use microbial fermentation to produce coenzyme Q10, the cost is high, so there is an urgent need for a production method that can increase production and reduce production costs
At present, there is no patent about enhancing the synthesis ability of coenzyme Q10 of Rhodobacter sphaeroides by knocking out the chlorophyll gene

Method used

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  • Coenzyme-Q10-production engineered bacteria construction method, engineered bacteria, and application thereof
  • Coenzyme-Q10-production engineered bacteria construction method, engineered bacteria, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1 produces the construction method of the engineering bacterium of coenzyme Q10

[0036] 1. Genomic DNA was extracted from Rhodobacter sphaeroides (all reagents used were from Biospin Bacterial Genomic DNA Extraction Kit)

[0037] 1. Absorb 0.5-4mL of Rhodobacter sphaeroides (up to 5×109 bacteria, said Rhodobacter sphaeroides were isolated from riverside sludge), centrifuge at 13,500rpm for 1 minute, and absorb the supernatant as much as possible.

[0038] 2. Add 100μL EL Buffer and pipette evenly with a tip.

[0039] 3. Incubate at 37°C for 40 minutes.

[0040] 4. Add 100μL RS Buffer, then add 10μL PK Solution, and mix well.

[0041] 5. Incubate at 56°C for 15 minutes, then remove.

[0042] 6. Add 200μL GA Buffer and mix well.

[0043] 7. Centrifuge at 12000 rpm for 1 minute. Transfer the supernatant to a new 1.5 mL centrifuge tube.

[0044] 8. Add 400μL of BA Buffer and mix well.

[0045] 9. Transfer the mixed liquid to the spin column. Centrifuge a...

Embodiment 2

[0126] The method of embodiment 2 application engineering bacteria production coenzyme Q10

[0127] First-class seeds: Pick a single clone of the NHU-ZAA strain and inoculate it in a 50-mL shake flask containing 10 mL of seed medium, at a rotation speed of 200 rpm, and incubate at 30°C for 23 hours.

[0128] Secondary seeds: transfer 1% of the first-grade seeds to a 50mL shake flask containing 20mL of seed medium, and cultivate them at 30°C and 200rpm for 23 hours to obtain the second-grade seeds.

[0129] Fermentation culture: inoculate 1% secondary seeds into 500mL containing 100mL fermentation medium, and culture at 30°C and 200rpm for 120h. Collect the bacterial fluid.

[0130] Among them, the seed medium contains per 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 PO 4 0.05g, MgSO 4 0.1g, FeSO 4 0.01g, CoCl 2 0.003g, MnSO 4 0.0001g, Vitamin B1 0.1μg, Vitamin K 0.1μg, Vitamin A 0.15...

experiment example

[0132] Experimental example HPLC is used to detect the comparison of the production of coenzyme Q10 produced by the strain without the chlorophyll gene and the NHU-ZAA strain, as shown in Table 1:

[0133] Table 1 Comparison of coenzyme Q10 production before and after strain transformation

[0134]

[0135] From the above table, the coenzyme Q10 output of the transformed strain increased from 2500mg / L to 2850mg / L.

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Abstract

The invention relates to the technical field of biology, and discloses a coenzyme-Q10-production engineered bacteria construction method, engineered bacteria, and an application thereof. The method comprises the steps that: a, total genomic DNA is extracted from Rhodobacter sphaeroides cacterial liquid; b, bchG homologous gene is obtained by amplification with a polymerase chain reaction; c, the amplified homologous gene is connected with broad-host plasmid, such that recombinant vector is constructed; d, the recombinant vector is transferred to Escherichia coli S17-1; and e, the Escherichia coli S17-1 is subjected to conjugal transfer with the Rhodobacter sphaeroides, such that the engineered bacteria with chlorophyll synthesis gene bchG knocked out are obtained. According to the method provided by the invention for improving coenzyme Q10 yield through knocking out Rhodobacter sphaeroides chlorophyll synthesis gene, coenzyme Q10 synthesis capacity can be improved by higher than approximately 15%. The method is suitable for coenzyme Q10 large-scale industrial production.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a construction method for producing coenzyme Q10 engineering bacteria, engineering bacteria and application methods. Background technique [0002] Coenzyme Q is a fat-soluble quinone compound that widely exists 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 (CoQ10). As 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 s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N15/09C12P7/66C12R1/01
Inventor 于洪巍陆文强石一军
Owner ZHEJIANG NHU CO LTD
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