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Gluconobacter oxydans engineering bacteria and preparation method and application thereof in preparing xylitol

A technology of Gluconobacter oxydans and a construction method, applied in the field of bioengineering, can solve the problems of low concentration and total amount of coenzyme NADH, low XDH activity, incomplete tricarboxylic acid cycle pathway, etc., achieving simple operation and solving transformation problems. low rate effect

Inactive Publication Date: 2013-03-20
NANJING UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Gluconobacter oxidans does not have a glycolytic pathway, and the tricarboxylic acid cycle pathway is not complete, which leads to a low concentration and total amount of reducing power and coenzyme NADH in the cell as a transfer phosphorylation electron donor, which in turn causes catalytic During the conversion process, the activity of XDH dependent on the coenzyme NADH is not high, and the conversion rate of xylitol is low

Method used

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  • Gluconobacter oxydans engineering bacteria and preparation method and application thereof in preparing xylitol
  • Gluconobacter oxydans engineering bacteria and preparation method and application thereof in preparing xylitol
  • Gluconobacter oxydans engineering bacteria and preparation method and application thereof in preparing xylitol

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Embodiment 1

[0039] Example 1: Construction of expression vectors.

[0040] P tufB , gox0145 gene sequence design primers to clone each gene fragment, the primer sequences used are shown in Table 1;

[0041] Table 1

[0042]

[0043] The reaction system is as follows: 10×PCR buffer5μL, Mg 2+ 4 μL, dNTP 4 μL, upstream and downstream primers 2 μL each, exTaq DNA polymerase 0.5 μL, genome template 1 μL, add ddH 2 O to a total reaction volume of 50 μL.

[0044] The PCR reaction conditions are: 95°C for 2min; 94°C for 30s; 55°C for 30s; 72°C for 1min; cycle 30 times; 72°C for 10min;

[0045] The cloned gene fragments and the extracted plasmid pBBR1MCS-5 were purified and recovered. Digest plasmid pBBR1MCS-5 and promoter fragment P with restriction enzymes KpnⅠ and XhoⅠ tufB , After the gel was cut and recovered, it was incubated with T4ligase at a constant temperature for the ligation reaction, and the resulting ligation solution was transformed into competent cells E.coli JM109, and t...

Embodiment 2

[0046] Embodiment 2: Transformation of recombinant engineered bacteria.

[0047] The transfer of recombinant plasmids between Escherichia coli and Gluconobacter oxydans (G. oxydans) can achieve conjugative transfer through the help of the plasmid pRK2013. The transformation method is called three-parent conjugation. (1) Bacteria culture: inoculate the donor bacteria (E.coli BL21 with recombinant plasmid), helper bacteria HB101 / pRK2013 in LB with corresponding antibiotics and culture for 8 hours, and acceptor bacteria NH-10 in the culture medium After culturing for 18 hours, conjugation experiments were carried out. (2) Three-parent conjugation: Take 1.5mL of the above-mentioned recipient parent bacterial solution, centrifuge to collect the bacterial cells, wash with normal saline twice, pour off the liquid, add 0.5mL of helper bacteria, mix well, centrifuge, add 1.5mL of donor bacteria and centrifuge , wash once with liquid medium, mix well, take a part of the liquid and appl...

Embodiment 3

[0048] Example 3: Fermentation and harvest of recombinant engineered bacteria.

[0049] Seed medium and slant medium (g / L): glucose 20, yeast powder 5, peptone 3; the amount of agar added to the solid medium was 1.5%. YPG medium (g / L): Glucose 30, yeast powder 10, sorbitol 10, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate trace (total 0.1~0.5). After adjusting the pH of the medium to 6.0-6.5, it was sterilized at 121°C; when the temperature of the medium used to cultivate recombinant bacteria dropped to 50-60°C, gentamicin and cephalosporin of corresponding concentrations were added.

[0050] Inoculate the activated strains G. oxydans NH-10 and G. oxydans pnadh-5 into the seed medium and incubate at a constant temperature for 18 hours until the middle and late logarithmic growth, and inoculate the fermentation medium at a ratio of 10% (v / v) Medium fermentation culture. Fermentation tank fermentation: YPG medium, on a 7.5L fermenter tank, th...

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Abstract

The invention discloses a preparation method of gluconobacter oxydans engineering bacteria, which performs over-expression of the key restriction enzyme glucose-6-phosphate dehydrogenase (G6PDH) for controlling the phosphopentose path to increase the metabolic flux of the path so as to realize cyclic regeneration of coenzyme NADH. The invention also discloses a fermentation technology of the engineering bacteria and application thereof in converting and preparing xylitol. According to the invention, the effect of the engineering bacteria in converting D-arabitol to generate xylitol is obviously superior to that of an original strain; and the conversion rate of xylitol against D-arabitol can reach 60-90%. The technology solves the problems of low conversion rate and low xylitol concentration of a bioconversion method for preparing xylitol to a certain degree, and has the advantages of economy and practicability, simplicity and convenience in operation and energy conservation and environmental protection.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a strain of gluconobacter oxydans engineering bacteria, a construction method thereof and an application in preparing xylitol. Background technique [0002] Xylitol has attracted more and more attention because its sweetness is comparable to that of sucrose, it can inhibit the activity of Proteus bacteria that form dental caries, its metabolism is not dependent on insulin, and it has functions such as lowering blood lipids and anti-ketone bodies. Xylitol is an excellent functional sweetener and an important platform compound, which has broad application prospects in food, medicine, national defense and light industry. It is predicted that the total demand for xylitol in the international market will reach more than 100,000 tons, and the market gap is as high as 40%, which shows that the market has great room for development. [0003] At present, xylitol is mainly produced b...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/53C12N15/63C12P19/02C12R1/01
Inventor 徐虹张金亮李莎冯小海
Owner NANJING UNIV OF TECH
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