CN103484417B - Gluconobacter oxydans improving 2-KLG fermentation yield and application thereof - Google Patents
Gluconobacter oxydans improving 2-KLG fermentation yield and application thereof Download PDFInfo
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Abstract
The invention discloses a gluconobacter oxydans for improving 2-KLG fermentation yield and application thereof. By means of a genetic engineering technology, pqqA and pqqABCDE genes derived from G. oxydans are respectively expressed in the one-step engineering bacterium G. oxydans that is constructed by the laboratory and used for producing 2-KLG from sorbitol. Accordingly, the dependence problem of small bacteria on associated bacteria is solved, direct conversion from D-sorbitol to 2-KLG is realized, and the vitamin C production process is simplified. In order to further improve the yield of 2-KLG, the key gene pqqA and the gene cluster pqqABCDE for synthesis of PQQ (pyrroloquinoline quinone) are respectively expressed in the G. oxydans one-step engineering bacterium, and the yield of 2-KLG is up to 51.8g/L, which is enhanced by 60% compared with the one-step engineering bacterium without expression of PQQ.
Description
Technical field
The present invention relates to a kind of gluconobacter suboxydans (Gluconobacter oxydans) engineering bacteria, a G.oxydans engineering bacteria particularly by regulating coenzyme metabolism to improve G.oxydans mono-step engineering bacteria production vitamin C precursor 2-KLG output, belongs to field of genetic engineering.
Background technology
Vitamins C (Vitamin C, VC), is also called 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, two-step fermenting produces ascorbic second step fermenting process, transform the process of 2-KLG by D-sorbose, the mixed fungus fermentation system be made up of bacillus megaterium (Bacillus megaterium) and ordinary student ketone group 2-KLG bacterium (Ketogulonigeniumvulgare) realizes.K.vulgare is Gram-negative bacteria, has related enzyme systems D-sorbose being converted into 2-KLG, but during its single culture, growth is extremely faint, accumulates 2-KLG hardly.B.megaterium is sporiferous gram-positive microorganism, does not participate in involved enzyme catalytic process directly.B.megaterium significantly can promote growth and the 2-KLG accumulation of K.vulgare.If removed from fermentation system by B.megaterium, the growth velocity of K.vulgare can significantly decline, and almost loses the ability of synthesis 2-KLG completely.
Although through practice in the industrial production for many years, utilize the mixed ascorbic technology of thalline system fermentative production comparatively ripe, but a series of problem that mixed thalline system causes still bring many restrictions to vitamins C industrial expansion: (1) strain improvement difficulty.Up to the present strain improvement normally using some phenotype of single bacterium as with reference to carrying out screening, and not yet finds suitable method for the operation of mixed fungus fermentation system.(2) metabolic engineering operation is difficult to carry out.Because the mono-bacteria growing of K.vulgare is very difficult, cause there is no suitable method at present and obtain targeted transformation.(3) complex manufacturing increases.In actual production process, rapid detection cannot be carried out to two bacterium ratios in mixed thalline system, and this ratio there is remarkably influenced to 2-KLG production.(4) Pollution of Phage problem.B.megaterium is extensively present in soil, all comparatively responsive to multiple phage.The tank switching caused due to phage problem be in vitamin C producing process one be difficult to the problem ignored.Vitamins C two-step fermentation genes involved is transformed into structure in G.oxydans by genetic engineering means and obtains one-step fermentation bacterial strain by this laboratory, realize generating 2-KLG by sorbyl alcohol through single bacterium one-step fermentation, relieve the problem that little bacterium relies on concomitance bacterium, simplify production of vitamin C technique.Participate in metabolism sorbyl alcohol and generate 3 key enzymes of 2-KLG all using pyrroloquinoline quinone (Pyrroloquinoline quinone, PQQ) as coenzyme, regulate coenzyme metabolism to be expected to the output improving 2-KLG further.
Coenzyme metabolic engineering G.oxydans mono-step bacterium is adopted to produce ascorbic acid biosynthesis precursor 2-KLG domestic there are no relevant report.
Summary of the invention
The object of this invention is to provide that a kind of 2-KLG output is improved G.oxydans mono-step engineering bacteria.
Described engineering bacteria contains pqqA, pqqABCDE gene, expression plasmid pGUC-tufB-sdh-GS-sndh in described G.oxydans mono-step bacterium.
Described pqqA, pqqABCDE gene nucleotide series is as shown in SEQ ID NO.1, SEQ ID NO.2.
On the plasmid pGUC1 that described pqqA, pqqABCDE gene clone builds in this laboratory.
Another technical problem that the present invention will solve is to provide a kind of construction process of G.oxydans genetic engineering bacterium of 2-KLG output increased.
For solving the problems of the technologies described above, concrete scheme of the present invention is:
1) according to this laboratory, pqqA, pqqABCDE gene order design primer annotated in the genome sequencing result of G.oxydans WSH-003 is cloned;
2) gene is connected with carrier obtains recombinant expression vector;
3) recombinant bacterial strain is obtained after the recombinant expression vector obtained being transformed G.oxydans.
Here is the specific descriptions of technical solution of the present invention:
The structure of plasmid and recombinant bacterium
PMD19-T order-checking is connected to after sdh and sndh annotated in the genome sequencing result of K.vulgare WSH-001 being merged by connection peptides GGGGS this laboratory, the strong promoter tufB deriving from G.oxydans is carried out amplification to be connected to cloning vector pMD19-T and to check order simultaneously, carry out double digestion after obtaining correct transformant and be connected to E.coli-G.oxydans shuttle vector pGUC1, obtain recombinant plasmid vector pGUC-tufB-sdh-GS-sndh.PqqA and pqqABCDE will annotated in the genome sequencing result of K.vulgare WSH-001 again, obtain gene respectively, be connected to cloning vector pMD19-T to check order, carry out after obtaining correct transformant double digestion be connected to this laboratory build G.oxydans mono-step bacterium in plasmid pGUC-tufB-sdh-GS-sndh on, build obtain pGUC-tufB-sdh-GS-sndh-pqqA, pGUC-tufB-sdh-GS
2-sndh-pqqABCDE, verifies positive transformant (occurring the band of 81bp and 3137bp respectively) by the recombinant expression vector Transformed E .coli JM109 built, bacterium colony PCR; To derive from the tufB promoter sequence amplification rear clone of G.oxydans WSH-003 to pMD19-T order-checking again, after obtaining correct transformant, double digestion is connected to pGUC-tufB-sdh-GS-sndh-pqqA, pGUC-tufB-sdh-GS
2-sndh-pqqABCDE, builds and obtains pGUC-tufB-sdh-GS-sndh-tufB-pqqA, pGUC-tufB-sdh-GS
2-sndh-tufB-pqqABCDE, after Transformed E .coliJM109, bacterium colony PCR verifies positive transformant (occurring the band of 490bp).By the method for triparental mating, recombinant expression vector is transferred in G.oxydans WSH-003 again.
The seed culture of recombinant bacterium and fermentation
Seed culture medium (g/L): sorbyl alcohol 15, yeast powder 1.0, pH 4.8 ~ 5.1, agar 20(solid medium), 121 DEG C 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 pH 5.1 ~ 5.4,121 DEG C of sterilizing 15min, penbritin final concentration 100 μ g/mL.
Culture condition: a few ring thalline of scraping is inoculated in 500mL two thorn shaking flask that 50mL liquid nutrient medium (adding final concentration 75 μ g/mL penbritin) is housed from solid plate, 30 DEG C of rotary shaker 200r/min shaking culture are to logarithmic phase (about 30h), the fresh culture of final concentration 75 μ g/mL penbritin is forwarded to by 15% (v/v) inoculum size, be cultured to logarithmic phase again, by 15% (v/v) inoculum size switching fermention medium, 30 DEG C, 200r/min, fermentation 168h.
Sorbyl alcohol, 2-KLG assay: liquid chromatography (LC)
Fermented sample moving phase ten times dilution, 0.45 μm of membrane filtration.Agilent 1100system, RioRad company AminexHPX-87H chromatographic column; Moving phase: 2.75 μm of ol/L vitriol oils; Column temperature: 35 DEG C; Flow velocity: 0.6mL/min; Sample size: 5 μ L; Detector: differential refraction detector.
The present invention is by genetic engineering modified, derive from pqqA and the pqqABCDE gene of G.oxydans, be expressed in the G.oxydans mono-step engineering bacteria by sorbyl alcohol production 2-KLG of this laboratory structure respectively, the output of 2-KLG brings up to 38.4g/L and 39.2g/L respectively, and a step engineering bacteria of not expressing PQQ improves 21%; Simultaneously containing in the G.oxydans mono-step engineering bacteria of pqqA and pqqABCDE, the output of 2-KLG can bring up to 51.8g/L, and a step engineering bacteria of not expressing PQQ improves 60%.
Embodiment
The structure of embodiment 1 expression vector
PMD19-T order-checking is connected to after sdh and sndh annotated in the genome sequencing result of K.vulgare WSH-001 being merged by connection peptides GGGGS this laboratory, the strong promoter tufB deriving from G.oxydans is carried out amplification to be connected to cloning vector pMD19-T and to check order simultaneously, carry out double digestion after obtaining correct transformant and be connected to E.coli-G.oxydans shuttle vector pGUC1, obtain recombinant plasmid vector pGUC-tufB-sdh-GS-sndh.According to this laboratory, pqqA and the pqqABCDE gene design primer annotated in the genome sequencing result of K.vulgareWSH-001 is increased again, check order after being connected respectively to cloning vector pMD19-T, carry out after obtaining correct transformant double digestion be connected to this laboratory build G.oxydans mono-step bacterium in plasmid pGUC-tufB-sdh-GS-sndh, build obtain pGUC-tufB-sdh-GS-sndh-pqqA and pGUC-tufB-sdh-GS
2-sndh-pqqABCDE, verifies positive transformant (occurring the band of 81bp and 3137bp respectively) by the recombinant expression vector Transformed E .coli JM109 built, bacterium colony PCR; Checked order to pMD19-T to the tufB promoter sequence amplification rear clone annotated in the genome sequencing result of G.oxydans WSH-003 in this laboratory, after obtaining correct transformant, double digestion is connected to pGUC-tufB-sdh-GS-sndh-pqqA, pGUC-tufB-sdh-GS again
2-sndh-pqqABCDE, builds and obtains expression vector pGUC-tufB-sdh-GS-sndh-tufB-pqqA and pGUC-tufB-sdh-GS
2-sndh-tufB-pqqABCDE.
The structure of embodiment 2 G.oxydans engineering bacteria
The expression vector built is transformed into E.coli JM109, be applied to (yeast extract paste 5g/L on the LB substratum containing penbritin, peptone 10g/L, NaCl 10g/L, solid medium adds 20g/L agar, 121 DEG C of sterilizing 15min), the transformant that picking transforms on rear plate carries out PCR checking, be transferred in G.oxydans WSH-003 by the method for triparental mating again, obtain 3 strain G.oxydans engineering bacterias.
Embodiment 3 fermentative production 2-KLG
Seed culture medium (g/L): sorbyl alcohol 15, yeast powder 1, pH 4.8 ~ 5.1, agar 20(solid medium), 121 DEG C 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 pH 5.1 ~ 5.4,121 DEG C of sterilizing 15min, penbritin final concentration 100 μ g/mL.
Culture condition: a few ring thalline of scraping is inoculated in 500mL two thorn shaking flask that 50mL liquid nutrient medium (adding final concentration 75 μ g/mL penbritin) is housed from solid plate, 30 DEG C of rotary shaker 200r/min shaking culture are to logarithmic phase (about 30h), the fresh culture of final concentration 75 μ g/mL penbritin is forwarded to by 15% (v/v) inoculum size, be cultured to logarithmic phase again, by 15% (v/v) inoculum size switching fermention medium, 30 DEG C, 200r/min, fermentation 168h.2-KLG output reaches 38.4g/L(pGUC-tufB-sdh-GS-sndh-tufB-pqqA respectively), 39.2g/L(pGUC-tufB-sdh-GS
2-sndh-tufB-pqqABCDE) and 51.8g/L(pGUC-tufB-sdh-GS-sndh-tufB-pqqA+pGUC-tufB-sdh-GS
2-sndh-tufB-pqqABCDE).
Although the present invention with preferred embodiment openly as above; but it is also not used 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, what therefore protection scope of the present invention should define with claims is as the criterion.
Claims (2)
1. the gluconobacter suboxydans improving 2-KLG fermentation yield (
gluconobacter oxydans) engineering bacteria, it is characterized in that by
pqqaBCDE, or
pqqa and
pqqaBCDE is expressed in simultaneously
g. oxydansin one step engineering bacteria, described in
g. oxydansexpression plasmid pGUC-in one step bacterium
tufB-
sdh-GS-
sndh, the building process of engineering bacteria is as follows:
Right
k. vulgareannotate in the genome sequencing result of WSH-001
sdhand
sndhbe connected to pMD19-T order-checking after being merged by connection peptides GGGGS, will derive from simultaneously
g. oxydansstrong promoter
tufBcarry out amplification to be connected to cloning vector pMD19-T and to check order, carry out double digestion after obtaining correct transformant and be connected to
e.coli-G.oxydansshuttle vector pGUC1, obtains recombinant plasmid vector pGUC-
tufB-
sdh-GS-
sndh;
Again will
k. vulgareannotate in the genome sequencing result of WSH-001
pqqa and
pqqaBCDE, obtains gene respectively, is connected to cloning vector pMD19-T and checks order, carry out double digestion and be connected to plasmid pGUC-after obtaining correct transformant
tufB-
sdh-GS-
sndhon, build and obtain pGUC-
tufB-
sdh-GS-
sndh-
pqqa, pGUC-
tufB-
sdh-GS
2-
sndh-
pqqaBCDE, transforms the recombinant expression vector built
e.colijM109, bacterium colony PCR verify positive transformant; To derive from again
g. oxydanswSH-003's
tufBpromoter sequence amplification rear clone is to pMD19-T order-checking, and after obtaining correct transformant, double digestion is connected to pGUC-
tufB-
sdh-GS-
sndh-
pqqa, pGUC-
tufB-
sdh-GS
2-
sndh-
pqqaBCDE, builds and obtains pGUC-
tufB-
sdh-GS-
sndh-
tufB-
pqqa, pGUC-
tufB-
sdh-GS
2-
sndh-
tufB-
pqqaBCDE, transforms
e.coliafter JM109, bacterium colony PCR verifies positive transformant, then is transferred to by recombinant expression vector by the method for triparental mating
g. oxydansin WSH-003;
Described
pqqa gene nucleotide series as SEQ ID NO.1, described in
pqqaBCDE gene nucleotide series is as shown in SEQ ID NO.2.
2. genetic engineering bacterium described in claim 1 is producing the application in 2-KLG.
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| CN104357347B (en) * | 2014-09-23 | 2017-08-11 | 山东鲁维制药有限公司 | One plant of Gluconobacter oxvdans and its application in fermentation production VC precursors |
| CN112899211B (en) * | 2021-03-23 | 2022-10-11 | 江南大学 | Method for increasing yield of 2-KLG in gluconobacter oxydans |
| 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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