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Genetic engineering bacterium of Gluconobacter oxydans (G.oxydans) and application thereof

A technology of gluconic acid bacteria and genetically engineered bacteria, applied in the biological field, can solve the problems of high oxygen consumption and no VHb, and achieve the effect of promoting growth and solving the problem of insufficient oxygen supply

Inactive Publication Date: 2012-07-11
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the protein has been successfully used in the antibiotic industry where oxygen consumption is large and dissolved oxygen is likely to become a limiting factor, high-density fermentation of genetically engineered bacteria, and animal and plant cell culture processes where oxygen supply is limited, but no VHb is oxidized. Reports of expression in Gluconobacter

Method used

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  • Genetic engineering bacterium of Gluconobacter oxydans (G.oxydans) and application thereof
  • Genetic engineering bacterium of Gluconobacter oxydans (G.oxydans) and application thereof
  • Genetic engineering bacterium of Gluconobacter oxydans (G.oxydans) and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, the construction of VHb expression vector

[0035] 1.1. Construction of plasmid pBBR-VHb

[0036] Primers were designed according to the nucleotide sequence of the VHb gene vgb announced in GenBank, and its sequence is as follows:

[0037] VHb-F: 5'-CGC TCTAGA AGGAAGACCCTCATGTTAG-3';

[0038] VHb-R: 5'-TTA CTCGAG CAAAGCCCAATTGGACG-3';

[0039] Using plasmid pBR322-vgb (Yang Lingchao, Sun Aiyou, etc., Journal of East China University of Science and Technology, 2006, 32 (8): 925-929) as template, with VHb-F and VHb-R as primers, PCR amplifies the target gene, and in XbaI and XhoI restriction sites (shown underlined) were introduced at both ends respectively, and the specific conditions are as follows:

[0040] Reaction system: 0.5 μl of Tag enzyme, 5 μl of 10×buffer, 1 μl of template, 4 μl of dNTP (2.5 mmol / L), 1.5 μl of each primer (10 μmol / L), ddH 2 O 36.5 μl;

[0041] Reaction process: 94°C for 5min, 94°C for 30s, 55°C for 45s, 72°C for 90s, 30 c...

Embodiment 2

[0053] Example 2, Expression and identification of VHb in the genetically engineered bacterium G.oxydans M5 pBBR-PtufB-VHb of Gluconobacter oxidans

[0054] 2.1. Culture method of G.oxydans M5 pBBR-PtufB-VHb

[0055] Medium formula (g / L): sorbitol 80, yeast powder 20, (NH 4 ) 2 8O 4 5. KH 2 PO 4 1.5, MgSO 4 ·7H 2 O 0.5, distilled water 1000mL. Culture is based on sterilization at 121°C for 20 minutes before use.

[0056] Pick a single colony of Gluconobacter oxydans genetically engineered bacteria from the sorbitol solid plate and inoculate it into a sorbitol medium test tube containing 25 μg / mL gentamicin sulfate, and culture it on a shaker at 30°C and 250rpm for about 20h as The seed solution was then transferred to a 500ml Erlenmeyer flask containing 100ml of the same medium at a ratio of 1%, and cultured with shaking at 250rpm and 30°C for 24h.

[0057] 2.2. Quantitative determination of VHb

[0058] The concentration of VHb was measured by carbon monoxide diff...

Embodiment 3

[0060] Embodiment 3, the growth of the genetically engineered bacteria of Gluconobacter oxidans under oxygen-limited and non-oxygen-limited culture conditions

[0061] The oxygen-limited and non-oxygen-limited culture of the genetically engineered bacteria of Gluconobacter oxydans is realized by different medium bottle volumes and shaker rotation speeds during culture. Filling 100ml medium in a 250ml Erlenmeyer flask and cultivating at 150rpm is defined as oxygen-limited culture; filling 50ml medium in a 250ml Erlenmeyer flask and cultivating at 250rpm is defined as non-oxygen-limited culture.

[0062] Cultivate seeds according to the method of 2.1, inoculate 1% of the inoculum into the above-mentioned Erlenmeyer flasks containing different amounts of sorbitol medium, and place them in a shaker at 30°C, 150rpm and 30°C, 250rpm, every 4h Take a sample once, dilute the OD value with fresh sorbitol medium to 0.2-0.6, use fresh sorbitol medium as a control, measure the absorbance ...

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Abstract

The invention discloses a recombinant plasmid for expressing vitreoscilla hemoglobin in a cell and a construction method of the recombinant plasmid, a genetic engineering bacterium of Gluconobacter oxydans (G.oxydans) and a construction method and application of the genetic engineering bacterium. The recombinant plasmid comprises a vitreoscilla hemoglobin gene vgb, a PtufB promoter of the Gluconobacter oxydans (G.oxydans) and a suitable carrier, wherein the genetic engineering bacterium is obtained by transforming recombinant plasmid into a host bacterium. The genetic engineering bacterium can be used for expressing the vitreoscilla hemoglobin in the cell, can be used for improving the yield of biomass and catalysate 1, 3-dihydroxyacetone under the condition of not changing the existing equipment and energy consumption pressure and provides an effective path for solving the problem of in sufficient oxygen supply in the cell cultivation and catalytic process of the Gluconobacter oxydans (G.oxydans).

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a genetically engineered bacterium of Gluconobacter oxydans (G.oxydans) and its application. Background technique [0002] 1,3-Dihydroxyacetone (1,3-Dihydroxyacetone, DHA) is the simplest polyhydroxy ketose, a white powdery crystal with sweet taste, easily soluble in water and organic substances such as ethanol, acetone and ether. solvent. The molecule of 1,3-dihydroxyacetone contains two hydroxyl groups and one ketone group. It has active chemical properties and widely participates in chemical reactions such as polymerization and condensation. It is an important intermediate and raw material for medicine and chemical synthesis. It is used in fine chemicals, food Various industries such as industrial and cosmetic industry play an important role. [0003] At present, the synthesis of 1,3-dihydroxyacetone can be realized through two ways, that is, chemical synthesis and m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/66C12N1/21C12N15/74C12P21/02C12P7/28C12R1/01
Inventor 林金萍魏东芝李明华吴建
Owner EAST CHINA UNIV OF SCI & TECH
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