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Gluconobacter oxydans engineering bacterium for producing sorbic ketone in high yield mode and construction method thereof

A technology of gluconic acid bacteria and glucose oxidation, applied in the field of genetic engineering, can solve the problems of difficult growth of small bacteria, difficulty in precise control, and many influencing factors, and achieve the effect of eliminating the dependence of associated bacteria, suitable for standardization, and good application prospects

Active Publication Date: 2014-11-26
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In 1972, Chinese scientists invented the vitamin C two-step fermentation method. Due to the greatly simplified process, greatly improved yield and safety, it was quickly promoted throughout the country. However, in the second-step mixed bacteria fermentation system, the implementation The microorganisms that transform sugar and acid are only small bacteria. It is difficult for small bacteria to grow alone. They need to be co-cultivated with large bacteria to grow normally. The process is complex, with many influencing factors, and it is difficult to control accurately. How to simplify the current two-step fermentation process is an urgent need. solved problem
[0003] There is no relevant report in China on the transformation of G.oxydans by genetic engineering technology to produce the intermediate product of vitamin C, sorbone

Method used

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  • Gluconobacter oxydans engineering bacterium for producing sorbic ketone in high yield mode and construction method thereof
  • Gluconobacter oxydans engineering bacterium for producing sorbic ketone in high yield mode and construction method thereof
  • Gluconobacter oxydans engineering bacterium for producing sorbic ketone in high yield mode and construction method thereof

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Experimental program
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Effect test

Embodiment 1

[0020] The construction of embodiment 1 expression vector

[0021] The sdh annotated in the whole genome sequencing results of K.vulgare WSH-001 in our laboratory and the promoter tufB of the elongation factor in G.oxydans WSH-003 were amplified and cloned into the pMD19-T vector, and positive transformants were picked Sequencing verification, the correct transformants were cultured to extract the plasmid, and the Gram-negative bacterial broad host shuttle plasmid vector pBBRMCS-2 was respectively digested with Xho I and Hind III and ligated, transformed into Escherichia coli (Escherichia coli) JM109, picked The transformant was cultured, and the extracted plasmid was verified by Xho I and Hind III digestion, and a 1858bp band appeared, which proved that the expression vector pBBRMCS-sdh had been successfully constructed. The sequenced correct pMD 19-T-tufB and pBBRMCS-sdh were digested with Kpn I and Xho I, then ligated, transformed into E.coli JM109, picked and cultured, and...

Embodiment 2

[0022] The construction of embodiment 2G.oxydans engineering bacteria

[0023] Culture the Escherichia coli recombinant strain E.coli / pBBRMCS-tufB-sdh constructed above, the auxiliary strain E.coli / pRK2013 (ATCC deposit number: 37159) and the recipient strain G.oxydans 621H to the logarithmic phase, press 2 Centrifuge after mixing at a ratio of 1:1, wash the cell pellet with normal saline, resuspend in 100 μL of normal saline, spread all the bacterial suspension on the sterile filter membrane of the sorbitol plate, incubate for 12 hours, and scrape the cell After appropriate dilution, apply to a selective plate with a final concentration of 50 μg / mL cefoxitin sodium and 50 μg / mL kanamycin, pick positive transfers for PCR verification, and extract the positive transfers after culture The plasmid was converted to E.coli JM109, and the extracted plasmid was verified by Kpn I, Xho I and Xho I, Hind III double enzyme digestion respectively, and bands of 1858bp and 500bp appeared, w...

Embodiment 3

[0024] Embodiment 3 fermentation produces sorbitol

[0025] Seed and slant medium (g / L): sorbitol 20, yeast extract 2, pH 4.8-5.1, agar 20 (slant medium), pH 7.0, sterilized at 121°C for 15 minutes, final concentration of kanamycin 50 μg / L mL.

[0026] Fermentation medium (g / L): Sorbitol 80, yeast extract 5, initial pH 5.1-5.4, sterilized at 121°C for 15 minutes, final concentration of kanamycin 50 μg / mL.

[0027] Culture conditions: inoculate the recombinant bacteria from the slant in 20mL seed medium, cultivate at 30°C and 200rpm for 24h, then inoculate in the fermentation medium with 15% inoculation amount, carry out shake flask fermentation at 30°C and 220rpm, and the fermentation period is 48h. Sorbitone yield was 72g / L.

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Abstract

The invention discloses a Gluconobacter oxydans engineering bacterium for producing vitamin C synthesis intermediate sorbic ketone and a construction method and application thereof, and belongs to the field of genetic engineering. By means of the genetic engineering technique, a sorbose dehydrogenase gene (sdh) derived from common ketogenic base Ketogulonigenium vulgare is cloned onto Gluconobacter oxydans to obtain the G. oxydans engineering bacterium for producing the sorbic ketone by utilizing sorbitol. G. oxydans WSH-003 which is provided by Jiangsu Jiangshan Pharmaceutical Co., Ltd is a fermented industrial strain obtained in a first step of a two-step fermentation method for producing 2-keto-L-gulonic acid (2-KLG). The sdh is cloned into the G. oxydans so that conversion from D-sorbitol to the vitamin C synthesis intermediate sorbic ketone is achieved, the foundation for further constructing the fermented industrial strain in one step to produce the direct precursor 2-KLG of vitamin C is laid, and the yield of the sorbic ketone is as high as 72g / L. Therefore, the Gluconobacter oxydans engineering bacterium has good application prospects.

Description

technical field [0001] The invention relates to a G.oxydans engineering bacterium with high sorbitol production and its construction method and application. The method of molecular biology is used to introduce the sorbose dehydrogenase (SDH) gene, so as to realize the production of sorbitol by metabolizing sorbitol, which belongs to genetic engineering field. Background technique [0002] Vitamin C (Vitamin C, VC), also known as L-ascorbic acid (L-ascorbic acid), is an important organic acid widely used in pharmaceutical, food, beverage, cosmetic and feed industries. Since no microorganisms that directly synthesize vitamin C have been found, the energy is mainly concentrated on the use of microbial fermentation to produce its intermediate products, especially its immediate precursor 2-keto-L-gulonic acid (2-keto-L-gulonic acid, 2-KLG). In 1972, Chinese scientists invented the vitamin C two-step fermentation method. Due to the greatly simplified process, greatly improved yi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74C12P7/26C12R1/01
Inventor 陈坚周景文高丽丽刘杰堵国成
Owner JIANGNAN UNIV
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