CN103484418A - Gluconobacter oxydans gene engineering bacteria for producing 2-KLG and its application - Google Patents

Abstract

The invention discloses a genetic engineering bacterium of Gluconobacter oxydans producing 2-KLG and its application. The invention uses genetic engineering technology to synthesize sorbose dehydrogenase ( SDH) and sorbone dehydrogenase (SNDH) genes were expressed in Gluconobacter oxydans after linking with peptides, and G.oxydans engineering bacteria that efficiently produced 2-KLG were obtained. G.oxydans is a commonly used strain in the first step of the fermentation process in the production of 2-KLG by the two-step fermentation method. Expressing the sdh and sndh genes in G.oxydans can relieve the problem of the dependence of the small bacteria on the accompanying bacteria and realize the transformation from D- The direct conversion of sorbitol to 2-KLG simplifies the production process of vitamin C, and the output of 2-KLG can reach 32.4g/L, which has a good application prospect.

Description

A kind of genetically engineered bacterium of Gluconobacter oxydans producing 2-KLG and its application

technical field

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

Vitamin C (Vitamin C, VC), also known as Ascorbic acid (Ascorbic acid), is an essential vitamin and antioxidant, widely used in industries such as medicine, food, feed and cosmetics. 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 in unstable production, and the tanks are often inverted due to the degradation of the bacteria, resulting in repeated heavy losses in production. The fermentation process of my country's vitamin C fermentation technology from sorbitol to 2-KLG involves three kinds of bacteria, which will inevitably cause a lot of waste of substrate and medium in the process of microbial metabolism. The fermentation process is divided into two steps, which not only prolongs the production cycle but also causes A huge waste of energy and manpower. Therefore, the existing vitamin C two-step fermentation process still has great potential for technological progress. G.oxydans is a commonly used strain in the first step of fermentation in the production of 2-KLG by the two-step fermentation method. Through genetic engineering, the genes related to the two-step fermentation of vitamin C are transformed into G.oxydans to construct a one-step fermentation strain, which realizes the production of sorbitol 2-KLG is produced by one-step fermentation of a single bacterium, which solves the problem of the dependence of small bacteria on associated bacteria and simplifies the production process of vitamin C.

There is no relevant report in China on the production of vitamin C synthetic precursor 2-KLG by using connecting peptide engineering to transform G.oxydans one-step bacteria.

Contents of the invention

The object of the present invention is to provide a 2-KLG-producing Gluconobacter oxydans engineering bacteria.

The engineering bacterium expresses sdh and sndh genes, and the sdh and sndh genes are connected through connecting peptides.

The nucleotide sequences of the sdh and sndh genes are shown as KVU-0203, whose NCBI accession number is AEM40042.1, and KVU-0095, whose NCBI accession number is AEM39934.1.

The sdh and sndh fusion genes are cloned on the G.oxydans-E.coli shuttle plasmid vector pGUC1.

Another technical problem to be solved by the present invention is to provide a method for constructing a 2-KLG-producing G.oxydans genetically engineered bacterium.

In order to solve the problems of the technologies described above, the specific solutions of the present invention are:

1) Design primers to clone sdh/sndh gene according to the gene sequence of sdh and sndh (NCBI accession numbers are AEM40042.1 and AEM39934.1) annotated in the whole genome sequencing results of K.vulgare WSH-001 in our laboratory or through chemical whole The gene is synthesized, and sdh and sndh are connected with different connecting peptides by fusion PCR;

2) Connect the sdh and sndh fusion genes to the vector to obtain a recombinant expression vector;

3) Transform the obtained recombinant expression vector into Gluconobacter oxydans to obtain a recombinant strain.

Below is the concrete description of technical scheme of the present invention:

Construction of plasmids and recombinant bacteria

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 then connected to the cloning vector pMD19-T for sequencing after fusion by PCR. 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 enzyme 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 -sndh, pGUC-tufB-sdh-EAK ₃ -sndh and pGUC-tufB-sdh-S ₃ N ₁₀ -sndh. The constructed recombinant expression vector was transformed into E.coli JM109, the positive transformant was verified by colony PCR (a band of about 3027bp appeared), and the recombinant expression vector was transferred to G.oxydansWSH-003 by the method of three-parent hybridization.

Seed culture and fermentation of recombinant bacteria

Seed medium (g/L): sorbitol 15, yeast powder 1.0, pH 4.8-5.1, agar 20 (solid medium), sterilized at 121°C for 15 minutes, final concentration of ampicillin 100 μg/mL.

Fermentation medium (g/L): sorbitol 15, yeast powder 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.

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, 30°C, 200r/min, and fermented for 168h.

Determination of sorbitol and 2-KLG content: liquid chromatography (LC)

Fermentation samples were diluted tenfold with mobile phase and filtered through a 0.45 μm filter membrane. Agilent1100system, Aminex HPX-87H chromatographic column of RioRad Company; mobile phase: 2.75 μmol/L concentrated sulfuric acid; column temperature: 35°C; flow rate: 0.6mL/min; injection volume: 5 μL; detector: differential refractive index detector.

In the present invention, sdh and sndh genes derived from Ketogulonigenium vulgare are expressed in oxidized G. oxydans WSH-003 after fusion with different linking peptides through genetic engineering transformation, and 10 strains using Sorbus Alcohol-producing 2-KLG engineered bacteria of G.oxydans. G.oxydans engineering bacteria are used to produce 2-KLG by one-step fermentation of sorbitol, which solves the problem of the dependence of small bacteria on accompanying bacteria and simplifies the production process of vitamin C. The output of 2-KLG can reach 34.8g/L, which has a good application prospect. The construction method provided by the invention is simple and suitable for standardization.

Description of drawings

Fig. 1 2-KLG yield after fusion of sorbose dehydrogenase and sorbone dehydrogenase by using 10 kinds of connecting peptides. G.oxydans/pGUC-t-sdh-GS-sndh(1), G.oxydans/pGUC-t-sdh-(GS) _2- sndh(2), G.oxydans/pGUC-t-sdh-(GS) _3- sndh(3), G.oxydans/pGUC-t-sdh-(PT) ₂ P-sndh(4), G.oxydans/pGUC-t-sdh-(PT) ₄ P-sndh(5), G .oxydans/pGUC-t-sdh-(PT) ₇ P-sndh(6), G.oxydans/pGUC-t-sdh-EAK-sndh(7), G.oxydans/pGUC-t-sdh-(EAK) ₂ -sndh (8), G.oxydans/pGUC-t-sdh-(EAK) ₃ -sndh (9), G.oxydans/pGUC-t-sdh-S ₃ N ₁₀ -sndh (10).

Detailed ways

The construction of embodiment 1 expression vector

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 After double enzyme digestion, it was connected to the E.coli-G.oxydans shuttle plasmid vector pGUC1, and 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 -sndh, pGUC-tufB-sdh-EAK ₃ -sndh and pGUC-tufB-sdh-S ₃ N ₁₀ -sndh.

The construction of embodiment 2G.oxydans engineering bacteria

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 15 min), 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.coliJM109, and then transferred to G.oxydans WSH- In 003, 10 strains of G.oxydans engineering bacteria were obtained.

Embodiment 3 fermentation produces 2-KLG

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.

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.

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, 30°C, 200r/min, and fermented for 168h. The highest yield of 2-KLG was 32.4g/L among the 10 engineered strains (Fig.1).

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

Claims (6)

1. a gluconobacter oxydans (Gluconobacter oxydans) genetically engineered bacterium producing 2-KLG is characterized in that expressing exogenous sdh, sndh gene, and described exogenous sdh, sndh gene are connected by connecting peptide. 2. The genetically engineered bacterium according to claim 1, characterized in that said sdh, sndh gene nucleotide sequence is shown in KVU-0203, NCBI accession number is AEM40042.1, and KVU-0095NCBI accession number is AEM39934.1. 3. The genetically engineered bacterium of Gluconobacter oxidans according to claim 1, characterized in that the connecting peptide is GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, PTPTP, PTPTPTPTP, PTPTPTPTPTPTPTP, EAAAK, EAAAKEAAAK, EAAAKEAAAKEAAAK or SSSNNNNNNNNNNNN. 4. the construction method of genetic engineering bacterium described in claim 1 is characterized in that comprising the steps: 1) According to the sdh (NCBI accession number: AEM40042.1) and sndh (NCBI accession number: AEM39934.1) gene sequences disclosed by this NCBI, primers were designed to clone or the sdh and sndh genes were synthesized by chemical synthesis, and the sdh and sndh genes were synthesized by fusion PCR. sndh connects with different connecting peptides; 2) connecting the fused gene to the vector to obtain a recombinant expression vector; 3) Transforming the obtained recombinant expression vector into Gluconobacter oxidans to obtain 2-KLG-producing Gluconobacter oxidans genetically engineered bacteria. 5. the application of the gluconobacter oxydans genetically engineered bacterium described in claim 1 in the fermentative production of 2-KLG. 6. The method according to claim 5, characterized in that the specific steps are: inoculate the genetically engineered bacterium of Gluconobacter oxydans in the 500mL double-thorned 500mL liquid culture medium (containing final concentration 75 μg/mL ampicillin) In a shaker flask, culture on a rotary shaker at 30°C at 200r/min until the logarithmic growth phase, transfer 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 right In the several growth period, transfer to the fermentation medium according to 15% (v/v) inoculation amount, 30°C, 200r/min, and ferment for 168h.

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