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Method for improving gluconobacter oxydans for producing 2-keto-L-gulconic acid

A technology for oxidizing glucose and acid bacteria, applied in the field of genetic engineering, can solve problems such as difficulty, prolong production cycle, pouring cans, etc., and achieve the effect of simple construction method and improved heat resistance

Inactive Publication Date: 2015-06-03
JIANGNAN UNIV
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  • Abstract
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  • Application Information

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Problems solved by technology

The mixed-bacteria fermentation control has added great difficulties to the production process, and the acid-producing properties of the small bacteria are unstable, and the tanks are often inverted due to the degradation of the bacteria, resulting in repeated severe damage to the production
The production of vitamin C in my country involves three kinds of bacteria, which will inevitably cause a lot of waste of substrate and medium in the process of microbial metabolism, and the two-step fermentation process not only prolongs the production cycle but also increases energy and labor costs.
Therefore, the existing vitamin C two-step fermentation process still has huge development potential

Method used

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  • Method for improving gluconobacter oxydans for producing 2-keto-L-gulconic acid
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  • Method for improving gluconobacter oxydans for producing 2-keto-L-gulconic acid

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Embodiment 1

[0033] The construction of embodiment 1 expression vector and one-step engineering bacterium

[0034] For the construction method of the Escherichia coli-Gluconobacter oxydans shuttle plasmid vector pGUC, please refer to the 2014 doctoral dissertation of Jiangnan University: Construction and optimization of engineering bacteria for the production of 2-keto-L-gulonic acid by single-step fermentation, Korea Lili.

[0035] (1) connecting the gene sdh encoding sorbose dehydrogenase and the gene sndh encoding sorbose dehydrogenase with the gene encoding the connecting peptide GGGGS to obtain the sequence of sdh-GGGGS-sndh as shown in SEQ ID NO.5; The sequence encoding the adapter protein SH3 (SEQ ID NO.2) was fused with sdh-GGGGS-sndh to obtain SH3-sdh-GGGGS-sndh; the plasmid pGUC was used as a vector, and the tufB (SEQ ID NO.4) promoter was ligated by enzyme digestion, The dehydrogenase fusion expression vector pGUC-tufB-SH3-sdh-GGGGS-sndh with adapter protein was obtained;

[00...

Embodiment 2

[0041] Embodiment 2 fermentation produces 2-KLG

[0042] Seed medium (g / L): sorbitol 15, yeast powder 1, pH 4.8-5.1, agar 20 (solid medium), sterilized at 121°C for 15 minutes, final concentration of ampicillin 100 μg / mL.

[0043] Fermentation medium (g / L): sorbitol 15, yeast extract 1.2, calcium chloride 0.2, initial pH 5.1-5.4, sterilized at 121°C for 15 minutes, final concentration of ampicillin 100 μg / mL.

[0044] Culture conditions: Scrape a few rings of bacteria from the solid plate and inoculate them in a 500mL double-thorn shaker flask filled with 50mL of liquid medium (added with a final concentration of 75μg / mL ampicillin), and culture on a rotary shaker at 30°C at 200r / min To the logarithmic growth phase (about 30h), according to the 15% (v / v) inoculation amount, transfer to the fresh medium of ampicillin with a final concentration of 75 μg / mL, and then culture to the logarithmic growth phase, according to 15% (v / v) ) inoculum amount was transferred to the fermenta...

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Abstract

The invention discloses a method for improving gluconobacter oxydans for producing 2-keto-L-gulconic acid, and belongs to the technical field of genetic engineering. On the basis of genetic engineering transformation, adaptor protein SH3 and ligand protein SH3lig of the adaptor protein, a CutA gene and a key enzyme gene in saccharic acid transformation are subjected to fusion expression, and G.oxydans is transformed to produce 2-KLG in a one-step fermentation manner, so that the yield of 2-KLG is finally increased to be 40.3 g / L, that is, the yield is increased by 24.4% when being compared with that of a one-step engineering bacterium of which CutA is not expressed; in addition, due to the thermal resistance of foreign protein, the thermal resistance of the engineering bacterium is further improved. Enzyme cross-linking inside cells can be achieved by expressing the foreign protein CutA, continuous catalytic reaction of a plurality of enzymes can be facilitated, and the method has significance in is one-step fermentation production of vitamin C.

Description

technical field [0001] The invention relates to a method for improving the production of 2-keto-L-gulonic acid by Gluconobacter oxidans, belonging to the technical field of genetic engineering. Background technique [0002] Vitamin C (Vitamin C, VC), also known as Ascorbic acid (Ascorbic acid), is an essential vitamin and antioxidant, widely used in medicine, food, feed and cosmetic industries. At present, the industrial production of vitamin C in China adopts a two-step fermentation method. The second step of this method is to convert sorbose into 2-KLG, which is composed of Bacillus megaterium and Ketogulonigenium vulgare. Realized by mixed bacteria fermentation system. In this system, only small bacteria (K.vulgare) are the microorganisms that perform sugar-acid conversion. However, small bacteria are difficult to grow alone and require the companionship of large bacteria (B.megaterium) to grow normally. The mixed-bacteria fermentation control has added great difficulti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12P7/60C12P17/04C12R1/01
Inventor 周景文陈坚王盼盼堵国成
Owner JIANGNAN UNIV
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