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Gluconobacter oxydans gene engineering bacteria for producing 2-KLG and its application

A technology of gluconic acid bacteria and genetically engineered bacteria, applied in the field of genetic engineering, achieving good application prospects, simple construction methods, and simplified production processes

Inactive Publication Date: 2014-01-01
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Using connecting peptide engineering to transform G.oxydans one-step bacteria to produce vitamin C synthetic precursor 2-KLG, there is no related report in China

Method used

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  • Gluconobacter oxydans gene engineering bacteria for producing 2-KLG and its application

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Experimental program
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Effect test

Embodiment 1

[0024] The construction of embodiment 1 expression vector

[0025] The sdh and sndh gene sequences annotated in the whole genome sequencing results of K. vulgare WSH-001 in our laboratory were connected with 10 kinds of connecting peptides (GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, PTPTP, PTPTPTPTP, PTPTPTPTPTPTPTP, EAAAK, EAAAKEAAAK, EAAAKEAAAKEAAAK and SSSNNNNNNNNNNNN) primers were amplified, and sequenced by connecting the cloning vector pMD19-T after PCR fusion. At the same time, the strong promoter tufB derived from G.oxydans was amplified and connected to the cloning vector pMD19-T for sequencing to obtain the correct transformation Afterwards, double digestion was performed and connected to the E.coli-G.oxydans shuttle plasmid vector pGUC1 to construct 10 fusion expression vectors pGUC-tufB-sdh-GS-sndh, pGUC-tufB-sdh-GS 2 -sndh, pGUC-tufB-sdh-GS 3 -sndh, pGUC-tufB-sdh-PT-sndh, pGUC-tufB-sdh-PT 4 -sndh, pGUC-tufB-sdh-PT 7 -sndh, pGUC-tufB-sdh-EAK-sndh, pGUC-tufB-sdh-EAK 2 ...

Embodiment 2

[0026] The construction of embodiment 2G.oxydans engineering bacteria

[0027]The constructed expression vector was transformed into E.coli JM109, spread on the LB medium containing ampicillin (yeast extract 5g / L, peptone 10g / L, NaCl10g / L, solid medium plus 20g / L agar, 121 ℃ sterilized for 15min), pick the transformant on the transformed plate for PCR verification, and a band of about 3027bp appears, which proves that it has been successfully transformed into E.coli JM109, and then transferred to G.oxydans WSH by the method of three-parent hybridization In -003, 10 strains of G.oxydans engineering bacteria were obtained.

Embodiment 3

[0028] Embodiment 3 fermentation produces 2-KLG

[0029] 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.

[0030] 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.

[0031] Culture conditions: Scrape a few rings of bacteria from the solid plate and inoculate them into a 500mL double-thorn shaker flask filled with 50mL of liquid medium (added with a final concentration of 75μg / mL ampicillin), and culture in a rotary shaker at 30°C at 200r / min When reaching the logarithmic growth phase (about 30h), transfer it to a fresh medium with a final concentration of 75 μg / mL ampicillin according to 15% (v / v) inoculation amount, and then culture until the logarithmic growth phase. ) inoculum amount was transferred to the fermentation medium, 3...

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Abstract

The invention discloses a Gluconobacter oxydans gene engineering bacteria for producing 2-KLG and its application. According to the invention, by means of a genetic engineering technology, sorbose dehydrogenase (SDH) and sorbosone dehydrogenase (SNDH) genes derived from Ketogulonigenium vulgare are connected by a connecting peptide and then expressed in Gluconobacter oxydans so as to obtain G. oxydans engineering bacteria for high efficiency production of 2-KLG. The G. oxydans is a strain commonly used in a first step fermentation process during two-step fermentation production of 2-KLG. The expression of sdh and sndh genes in G.oxydans can dissolve the dependence problem of small bacteria on associated bacteria, thus realizing direct conversion from D-sorbitol to 2-KLG, and simplifying the vitamin C production process. With a 2-KLG yield of 32.4g / L, the Gluconobacter oxydans gene engineering bacteria has very good application prospects.

Description

technical field [0001] The invention relates to an engineering bacterium of Gluconobacter oxydans and its application, in particular to an engineering bacterium of Gluconobacter oxydans producing 2-KLG and its application, belonging to the 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. In the second-step mixed-bacteria fermentation system, only small bacteria perform sugar-acid conversion. It is difficult for small bacteria to grow alone, and they need to be co-cultured with large bacteria to grow normally. The fermentation control of the two kinds of bacteria has added great difficulties to the production process, and the acid-producing properties of the small bacteria are unstable, resulting ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12P7/60C12R1/01
Inventor 陈坚周景文高丽丽堵国成
Owner JIANGNAN UNIV
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