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Combination gene of gene cluster ABCDEN by synthesizing sorbose dehydrogenase genes, sorbosone dehydrogenase genes and pyrroloquinoline quinone

A technology of sorbose dehydrogenase and sorbone dehydrogenase, which is applied in microorganism-based methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve problems such as weak acid production capacity, and achieve the effect of increasing yield

Inactive Publication Date: 2012-12-12
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, Gluconobacter oxydans is an acid-producing bacterium, but its ability to grow and produce acid is weak when cultured alone, and it needs to be promoted by the companion bacteria Bacillus megaterium to complete the process of growth and acid production
There is still room for improvement in the efficiency of mixed fermentation of the two bacteria to produce 2-keto-L-gulonic acid, and improving the sugar-acid conversion ability of the acid-producing bacteria Gluconobacter oxidans is the key to improving the production efficiency of mixed bacteria fermentation

Method used

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  • Combination gene of gene cluster ABCDEN by synthesizing sorbose dehydrogenase genes, sorbosone dehydrogenase genes and pyrroloquinoline quinone
  • Combination gene of gene cluster ABCDEN by synthesizing sorbose dehydrogenase genes, sorbosone dehydrogenase genes and pyrroloquinoline quinone
  • Combination gene of gene cluster ABCDEN by synthesizing sorbose dehydrogenase genes, sorbosone dehydrogenase genes and pyrroloquinoline quinone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] (1) Construction of Gluconobacter oxidans combining sorbose dehydrogenase gene with pyrroloquinoline quinone synthesis gene cluster ABCDEN

[0024] With the Gluconobacter oxydans genome as a template, with sequence listing SEQ ID No.7 and SEQ ID No.8 as primers, PCR amplifies the sorbose dehydrogenase gene with KpnI and HindIII restriction sites (sequence listing SEQ ID No.1); using SEQ ID No.11 and SEQ ID No.12 as primers, using the Gluconobacter oxidans genome as a template, PCR amplified the pyrroloquinoline quinone synthetic gene cluster ABCDEN with SpeI and SacI restriction sites and its upstream promoter (SEQ ID No.3 in the sequence table); after double-enzyme digestion, it was connected to the vector pBBR1MCS digested with KpnI and SacI, and the ligation product was transformed into E. coli DH5α to verify the correct plasmid connection, and then electro- The transformation method introduces the Gluconobacter oxidans into the Gluconobacter oxidans which combines t...

Embodiment 2

[0030] Mixed fermentation of Gluconobacter oxydans and Bacillus megaterium containing recombinant vector:

[0031] (1) Solid culture:

[0032] The preparation of the solid medium is as follows: Weigh 20g of L-sorbose, 3g of corn steep liquor, 3g of beef extract, 3g of yeast extract powder, 1g of urea, 10g of peptone, 20g of agar, KH 2 PO 4 1g, MgSO 4 0.2g, CaCO 3 Add 1 g of water to 1 L, adjust the pH to 6.8, and sterilize at 121°C for 20 minutes to make a solid medium;

[0033] Inoculate 150 μL of the three recombinant bacteria and the original strain Gluconobacter oxidans obtained in Example 1 and preserved in 20% aqueous glycerol solution on solid medium, and incubate at 30°C for 48 hours; take 150 μL and store in volume Bacillus megaterium in 20% glycerol aqueous solution was inoculated on solid medium and cultured at 30°C for 24 hours;

[0034] (2) Seed cultivation:

[0035] The preparation of the seed medium is as follows: weigh 20g of L-sorbose, 3g of corn stee...

Embodiment 3

[0042] Determination of 2-keto-L-gulonic acid and L-sorbose content:

[0043] Using high performance liquid chromatography (HPLC).

[0044] Sample preparation: Take 1 mL of the fermentation broth fermented for different times in a 1.5 mL centrifuge tube, centrifuge at 10,000 r / min for 3 min, take 100 μL of the supernatant in a 1.5 mL centrifuge tube, and add 900 μL of mobile phase (5 mM H 2 SO 4 ) to obtain ten-fold diluted samples. After shaking and mixing, filter the sample with a 0.22 μm cellulose microporous membrane to obtain the sample to be tested.

[0045] High performance liquid chromatography conditions: chromatographic column: bio-rad HPX-87H, mobile phase: 5mM H 2 SO 4 , flow rate: 0.6mL / min, column temperature: 65°C, differential detector.

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Abstract

The invention discloses a combination gene of a gene cluster ABCDEN by synthesizing sorbose dehydrogenase genes, sorbosone dehydrogenase genes and pyrroloquinoline quinone and relates to a SEQ ID No. 6 nucleotide sequence in a sequence table. According to the method, the yield of 2-keto-L-gulonic acids in mixed cultivation with bacillus megatherium can be improved apparently by constructing gluconobacter oxydans recombinant bacteria of different combination modes of the gene cluster ABCDEN synthesized by the sorbose dehydrogenase genes, the sorbosone dehydrogenase genes and the pyrroloquinoline quinine, and the yield can be improved as high as 20%.

Description

technical field [0001] The invention belongs to the field of industrial microorganisms, and relates to a combination gene of sorbose dehydrogenase gene, sorbone dehydrogenase gene and pyrroloquinoline quinone synthetic gene cluster ABCDEN. Background technique [0002] Vitamin C (Vc), also known as ascorbic acid, is a trace water-soluble vitamin necessary for body nutrition and growth. It plays an important role in anti-oxidation and maintaining metabolic balance. It is widely used in the pharmaceutical industry, food industry, and cosmetics industry. And the feed industry, etc., its application scope is continuously expanding, and the market demand is stable. [0003] In industrial production, vitamin C is mainly produced through the "two-step fermentation method", that is, the first step of fermentation uses Acetobacter black to convert D-sorbitol into L-sorbose, and the second step of fermentation uses Gluconobacter oxydans (Gluconobacter oxydans ) and Bacillus megateriu...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N1/21C12R1/01
Inventor 元英进杜瑾
Owner TIANJIN UNIV
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