CN103484418A - Gluconobacter oxydans gene engineering bacteria for producing 2-KLG and its application - Google Patents
Gluconobacter oxydans gene engineering bacteria for producing 2-KLG and its application Download PDFInfo
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- CN103484418A CN103484418A CN201310466179.5A CN201310466179A CN103484418A CN 103484418 A CN103484418 A CN 103484418A CN 201310466179 A CN201310466179 A CN 201310466179A CN 103484418 A CN103484418 A CN 103484418A
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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
The present invention relates to a kind of oxidizing glucose acidfast bacilli engineering bacteria and application thereof, particularly a kind of oxidizing glucose acidfast bacilli engineering bacteria and application thereof of producing 2-KLG, belong to field of genetic engineering.
Background technology
Vitamins C (Vitamin C, VC), be called again xitix (Ascorbic acid), is a kind of VITAMIN and antioxidant of needed by human, is widely used in the industry such as medicine, food, feed and makeup.Current domestic industrial vitamin C is produced and is adopted two-step fermenting, in second step mixed fermentation system, carries out the microorganism that saccharic acid transforms and only has little bacterium, and little bacterium growth separately is very difficult, need to could normal growth with large bacterium co-cultivation.The fermentations of two kinds of bacterium is controlled as production technique has increased very large difficulty, and little bacterium to produce acid shape unstable, thereby cause producing unstable, often because spawn degeneration causes tank switching, cause suffering heavy losses repeatedly in production.The fermenting process of China's vitamin c fermenting technology from the sorbyl alcohol to 2-KLG participated in by 3 kinds of bacteriums, certainly will in microbial metabolism, cause a large amount of wastes of substrate and substratum, fermenting process is divided into two steps and has not only extended the production cycle and also cause a large amount of wastes of the energy and manpower.Therefore, existing vitamins C two-step fermentation technique remains the potentiality in huge technical progress.G.oxydans is that two-step fermenting is produced the first step fermenting process bacterial classification commonly used in 2-KLG, by genetic engineering means, vitamins C two-step fermentation related gene is transformed in G.oxydans and builds and obtain the one-step fermentation bacterial strain, realization generates 2-KLG by sorbyl alcohol through single bacterium one-step fermentation, remove the problem that little bacterium relies on concomitance bacterium, simplified production of vitamin C technique.
Adopt the engineered G.oxydans mono-step bacterium of connection peptides to produce the synthetic precursor 2-KLG of vitamins C domestic there are no relevant report.
Summary of the invention
The oxidizing glucose acidfast bacilli engineering bacteria that the purpose of this invention is to provide a kind of 2-KLG of production.
Described engineering bacterium expression sdh, sndh gene, sdh, sndh gene are connected by connection peptides.
Described sdh, sndh gene nucleotide series are AEM40042.1 as KVU-0203, the NCBI number of logging in, and the KVU-0095NCBI number of logging in is shown in AEM39934.1.
Described sdh, sndh fusion gene cloning are on G.oxydans-E.coli shuttle vector pGUC1.
Another technical problem that the present invention will solve is to provide the construction process of the G.oxydans genetic engineering bacterium of a kind of 2-KLG of production.
For solving the problems of the technologies described above, concrete scheme of the present invention is:
1) sdh annotated in the genome sequencing result to K.vulgare WSH-001 according to this laboratory, the sndh(NCBI number of logging in are AEM40042.1, AEM39934.1) gene order design primer clones the sdh/sndh gene or, by chemical complete synthesis acquisition gene, by fusion, PCR is connected with sndh sdh with different connection peptides;
2) sdh, sndh fusion gene are connected and obtain recombinant expression vector with carrier;
3) will after the recombinant expression vector conversion oxidizing glucose acidfast bacilli (Gluconobacter oxydans) obtained, obtain recombinant bacterial strain.
Below the specific descriptions of technical solution of the present invention:
The structure of plasmid and recombinant bacterium
The sdh annotated in genome sequencing result by this laboratory to K.vulgare WSH-001 and sndh gene order are respectively to be connected with 10 kinds of connection peptides (GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, PTPTP, PTPTPTPTP, PTPTPTPTPTPTPTP, EAAAK, EAAAKEAAAK, EAAAKEAAAKEAAAK and SSSNNNNNNNNNN) primer increased, be connected to cloning vector pMD19-T order-checking after merging by PCR, the strong promoter tufB that simultaneously will derive from G.oxydans increases and is connected to cloning vector pMD19-T order-checking, obtain after correct transformant and carry out double digestion and be connected to E.coli-G.oxydans shuttle vector pGUC1, structure obtains 10 fusion expression vector 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
3n
10-sndh.By the recombinant expression vector Transformed E .coli JM109 built, bacterium colony PCR verifies positive transformant (band that about 3027bp occurs), and the method by triparental mating is transferred to recombinant expression vector in G.oxydans WSH-003.
The seed culture of recombinant bacterium and fermentation
Seed culture medium (g/L): sorbyl alcohol 15, yeast powder 1.0, pH4.8～5.1, agar 20(solid medium), 121 ℃ of sterilizing 15min, penbritin final concentration 100 μ g/mL.
Fermention medium (g/L): sorbyl alcohol 15, yeast powder 1.2, calcium chloride 0.2, initial pH5.1～5.4,121 ℃ of sterilizing 15min, penbritin final concentration 100 μ g/mL.
Culture condition: from solid plate, a few ring thalline of scraping are inoculated in the two thorn of the 500mL shaking flask that 50mL liquid nutrient medium (adding final concentration 75 μ g/mL penbritins) is housed, 30 ℃ of rotary shaking table 200r/min shaking culture are to logarithmic phase (30h left and right), be forwarded to the fresh culture of final concentration 75 μ g/mL penbritins by 15% (v/v) inoculum size, be cultured to again logarithmic phase, by 15% (v/v) inoculum size switching fermention medium, 30 ℃, 200r/min, fermentation 168h.
Sorbyl alcohol, 2-KLG assay: liquid chromatography (LC)
Ten times of dilutions of moving phase for fermented sample, 0.45 μ m membrane filtration.Agilent1100system, the Aminex HPX-87H of RioRad company chromatographic column; Moving phase: the 2.75 μ mol/L vitriol oils; Column temperature: 35 ℃; Flow velocity: 0.6mL/min; Sample size: 5 μ L; Detector: differential refraction detector.
The present invention is by genetic engineering modified, sdh, the sndh gene that will derive from ordinary student ketone group 2-KLG bacterium (Ketogulonigenium vulgare) are expressed in oxidation G.oxydans WSH-003 after merging with different connection peptides, have obtained 10 strains and have utilized sorbyl alcohol to produce the G.oxydans engineering bacteria of 2-KLG.Adopt the G.oxydans engineering bacteria to utilize the sorbyl alcohol one-step fermentation to produce 2-KLG, removed the problem that little bacterium relies on concomitance bacterium, simplified production of vitamin C technique.The output of 2-KLG can reach 34.8g/L, has good application prospect.Construction process provided by the invention is simple, is suitable for stdn.
The accompanying drawing explanation
Fig. 1 adopts 10 kinds of connection peptides that sorbose dehydrogenase and sorbic ketone dehydrogenase are merged to rear 2-KLG output.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)
2P-sndh(4),G.oxydans/pGUC-t-sdh-(PT)
4P-sndh(5),G.oxydans/pGUC-t-sdh-(PT)
7P-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
3N
10-sndh(10)。
Embodiment
The structure of embodiment 1 expression vector
The sdh annotated in genome sequencing result by this laboratory to K.vulgare WSH-001 and sndh gene order are respectively to be connected with 10 kinds of connection peptides (GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, PTPTP, PTPTPTPTP, PTPTPTPTPTPTPTP, EAAAK, EAAAKEAAAK, EAAAKEAAAKEAAAK and SSSNNNNNNNNNN) primer increased, connecting cloning vector pMD19-T after merging by PCR is checked order, the strong promoter tufB that simultaneously will derive from G.oxydans increases and is connected to cloning vector pMD19-T order-checking, obtain after correct transformant and carry out double digestion and be connected to E.coli-G.oxydans shuttle vector pGUC1, be building up to 10 fusion expression vector 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
3n
10-sndh.
The structure of embodiment 2G.oxydans engineering bacteria
The expression vector built is transformed into to E.coli JM109, be applied to (yeast extract paste 5g/L on the LB substratum that contains penbritin, peptone 10g/L, NaCl10g/L, solid medium adds 20g/L agar, 121 ℃ of sterilizing 15min), the transformant that picking transforms on rear plate carries out the PCR checking, the band that about 3027bp occurs, proof successfully is transformed in E.coli JM109, method by triparental mating is transferred in G.oxydans WSH-003 again, obtains 10 strain G.oxydans engineering bacterias.
Seed culture medium (g/L): sorbyl alcohol 15, yeast powder 1, pH4.8～5.1, agar 20(solid medium), 121 ℃ of sterilizing 15min, penbritin final concentration 100 μ g/mL.
Fermention medium (g/L): sorbyl alcohol 15, yeast extract paste 1.2, calcium chloride 0.2, initial pH5.1～5.4,121 ℃ of sterilizing 15min, penbritin final concentration 100 μ g/mL.
Culture condition: from solid plate, a few ring thalline of scraping are inoculated in the two thorn of the 500mL shaking flask that 50mL liquid nutrient medium (adding final concentration 75 μ g/mL penbritins) is housed, 30 ℃ of rotary shaking table 200r/min shaking culture are to logarithmic phase (30h left and right), be forwarded to the fresh culture of final concentration 75 μ g/mL penbritins by 15% (v/v) inoculum size, be cultured to again logarithmic phase, by 15% (v/v) inoculum size switching fermention medium, 30 ℃, 200r/min, fermentation 168h.Obtaining the 2-KLG production peak in 10 strain engineering bacterias is 32.4g/L (Fig.1).
Although the present invention with preferred embodiment openly as above; but it is not in order to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, so protection scope of the present invention should be with being as the criterion that claims were defined.
Claims (6)
1. oxidizing glucose acidfast bacilli (Gluconobacter oxydans) genetic engineering bacterium of producing 2-KLG, is characterized in that expressing external source sdh, sndh gene, and described external source sdh, sndh gene connect by connection peptides.
2. genetic engineering bacterium claimed in claim 1, is characterized in that described sdh, sndh gene nucleotide series are AEM40042.1 as KVU-0203, the NCBI number of logging in, and the KVU-0095NCBI number of logging in is shown in AEM39934.1.
3. Gluconobater oxydans genetic engineering strain claimed in claim 1, is characterized in that described connection peptides is GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, PTPTP, PTPTPTPTP, PTPTPTPTPTPTPTP, EAAAK, EAAAKEAAAK, EAAAKEAAAKEAAAK or SSSNNNNNNNNNN.
4. the construction process of the described genetic engineering bacterium of claim 1, is characterized in that comprising the steps:
1) clone or adopt chemical complete synthesizing process sdh, sndh gene according to the disclosed sdh of this NCBI (the NCBI number of logging in is AEM40042.1), sndh (the NCBI number of logging in is AEM39934.1) gene order design primer, by merging PCR, sdh is connected with different connection peptides with sndh;
2) gene after merging is connected and obtains recombinant expression vector with carrier;
3) Gluconobater oxydans genetic engineering strain of 2-KLG will be obtained producing after the recombinant expression vector conversion oxidizing glucose acidfast bacilli obtained.
5. the application of the described Gluconobater oxydans genetic engineering strain of claim 1 in fermentative production 2-KLG.
6. method according to claim 5, it is characterized in that concrete steps are: described Gluconobater oxydans genetic engineering strain is inoculated in the two thorn of the 500mL shaking flask that 50mL liquid nutrient medium (containing final concentration 75 μ g/mL penbritins) is housed, 30 ℃ of rotary shaking table 200r/min shaking culture are to logarithmic phase, be forwarded to the fresh culture of final concentration 75 μ g/mL penbritins by 15% (v/v) inoculum size, be cultured to again logarithmic phase, by 15% (v/v) inoculum size switching fermention medium, 30 ℃, 200r/min, fermentation 168h.
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Cited By (7)
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|---|---|---|---|---|
| CN104357347A (en) * | 2014-09-23 | 2015-02-18 | 虞龙 | Gluconobacter oxydans and application of gluconobacter oxydans in production of VC precursor by fermentation |
| CN104357529A (en) * | 2014-10-15 | 2015-02-18 | 沈阳药科大学 | Method for improving production capacity of 2-KGA (2-keto-L-gulonic acid) through enhancement of Ketogulonogeniumvulgarum carbon metabolism level |
| CN104673736A (en) * | 2015-03-11 | 2015-06-03 | 江南大学 | Method for improving gluconobacter oxydans for producing 2-keto-L-gulconic acid |
| CN106282084A (en) * | 2016-09-29 | 2017-01-04 | 江南大学 | A kind of genetic engineering bacterium of the Gluconobacter oxvdans producing 2 ketone group L 2-KLGs |
| CN108342405A (en) * | 2017-01-23 | 2018-07-31 | 复旦大学 | IL21 fusion proteins and preparation method thereof and the purposes in preparing targeting therapy on tumor drug |
| CN112111437A (en) * | 2020-05-25 | 2020-12-22 | 江南大学 | Recombinant bacillus subtilis with improved 2' -fucosyllactose yield and construction method thereof |
| CN114480236A (en) * | 2022-02-23 | 2022-05-13 | 江南大学 | Construction method of 2-KLG (bacillus gluconicum) production one-step strain chassis cell bank |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104357347A (en) * | 2014-09-23 | 2015-02-18 | 虞龙 | Gluconobacter oxydans and application of gluconobacter oxydans in production of VC precursor by fermentation |
| CN104357529A (en) * | 2014-10-15 | 2015-02-18 | 沈阳药科大学 | Method for improving production capacity of 2-KGA (2-keto-L-gulonic acid) through enhancement of Ketogulonogeniumvulgarum carbon metabolism level |
| CN104673736A (en) * | 2015-03-11 | 2015-06-03 | 江南大学 | Method for improving gluconobacter oxydans for producing 2-keto-L-gulconic acid |
| CN106282084A (en) * | 2016-09-29 | 2017-01-04 | 江南大学 | A kind of genetic engineering bacterium of the Gluconobacter oxvdans producing 2 ketone group L 2-KLGs |
| CN106282084B (en) * | 2016-09-29 | 2019-07-02 | 江南大学 | A kind of genetic engineering bacterium for the Gluconobacter oxvdans producing 2-keto-L-gulonic acid |
| CN108342405A (en) * | 2017-01-23 | 2018-07-31 | 复旦大学 | IL21 fusion proteins and preparation method thereof and the purposes in preparing targeting therapy on tumor drug |
| CN112111437A (en) * | 2020-05-25 | 2020-12-22 | 江南大学 | Recombinant bacillus subtilis with improved 2' -fucosyllactose yield and construction method thereof |
| CN112111437B (en) * | 2020-05-25 | 2023-09-05 | 江南大学 | Recombinant bacillus subtilis with improved yield of 2' -fucosyllactose and construction method thereof |
| CN114480236A (en) * | 2022-02-23 | 2022-05-13 | 江南大学 | Construction method of 2-KLG (bacillus gluconicum) production one-step strain chassis cell bank |
| CN114480236B (en) * | 2022-02-23 | 2023-10-03 | 江南大学 | Construction method of one-step bacillus gluconate chassis cell bank for producing 2-KLG |
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