CN110643651B - Method for promoting expression of deoxyviolacein in chromobacterium violaceum - Google Patents
Method for promoting expression of deoxyviolacein in chromobacterium violaceum Download PDFInfo
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- CN110643651B CN110643651B CN201910938856.6A CN201910938856A CN110643651B CN 110643651 B CN110643651 B CN 110643651B CN 201910938856 A CN201910938856 A CN 201910938856A CN 110643651 B CN110643651 B CN 110643651B
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- deoxyviolacein
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- schiff base
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
- C12P17/165—Heterorings having nitrogen atoms as the only ring heteroatoms
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
- C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
- C07D233/64—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to a method for promoting the expression of deoxyviolacein in chromobacterium violaceum, belonging to the field of biological medicine. The main technical scheme is as follows: culturing histidine fatty aldehyde Schiff base at 25-35 deg.C with activated Chromobacterium violaceum for 12-48 hr, and collecting deoxyviolacein expressed by thallus. The histidine fatty aldehyde Schiff base provided by the invention has a five-membered ring head structure and a long-chain fatty aldehyde tail part which are very similar to a signal molecule n-hexane-L-homoserine lactone of a chromobacterium violaceum quorum sensing system, and can effectively promote the expression of deoxyviolacein in chromobacterium violaceum.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to a method for promoting expression of deoxyviolacein in chromobacterium violaceum.
Background
Violacein is a microbial metabolite belonging to the indole derivatives and is a water insoluble bluish-black pigment. In the 19 th century, it was found that Chromobacterium violacea expressed violacein, and thereafter, various species were successively found to produce violacein. Subsequently, it was found that the blue-violet pigment has a lower content of deoxyviolacein in addition to violacein.
Research shows that violacein has very excellent biological activity, including broad-spectrum antibacterial property (effectively inhibiting streptococcus, bacillus, staphylococcus aureus, pseudomonas and the like); antiviral property (activity against herpes simplex virus and poliovirus infecting HeLa cells); anti-tumor cells (significantly inhibiting colon cancer HT29 cell proliferation); genotoxicity and antiprotozoal activity, etc. Deoxyviolacein is different from violacein in structure by one hydroxyl group, and the expression quantity of the pigment in thalli is smaller and is only one tenth of that of violacein, but the deoxyviolacein also has the activities of resisting tumors, gram-positive bacteria, plant pathogenic bacteria and the like. However, the two blue-violet microbial metabolites are currently extremely low in yield, and cannot be industrially produced. Therefore, a method for promoting the expression of violacein and deoxyviolacein in chromobacterium violaceum is urgently needed to solve the problem of low yield.
The expression of violacein and deoxyviolacein is regulated by the CviI/CviR quorum sensing system in Chromobacterium violaceum. The main signal molecule of the system is N-hexane-L-homoserine lactone (HHL). The molecular structural formula is as follows.
After the HHL signal molecule is combined with the receptor protein CviR, the intracellular domain of the receptor protein is phosphorylated, and then a downstream signal path is activated to realize the expression of the deoxyviolacein. If the signal molecule intensity can be enhanced in the extracellular environment, the signal of the whole channel is expected to be enhanced, and the expression quantity of the deoxyviolacein is increased.
Disclosure of Invention
In order to solve the problems in the prior art, the invention designs and synthesizes histidine fatty aldehyde Schiff base, which comprises histidine heptanal, histidine octanal, histidine decanal and histidine dodecanal. The compounds show excellent biological activity in promoting the expression of deoxyviolacein in chromobacterium violaceum. The invention provides a method for promoting expression of deoxyviolacein in chromobacterium violaceum, which effectively improves the yield of the deoxyviolacein and provides a theoretical basis for realizing industrial mass production of the deoxyviolacein.
The technical scheme of the invention is as follows: a method for promoting the expression of deoxyviolacein in Chromobacterium violaceum, comprising the following steps:
co-culturing histidine fatty aldehyde Schiff base with certain concentration and activated chromobacterium violaceum at 25-35 deg.c for 12-48 hr, and collecting deoxyviolacein expressed by thallus;
the preparation method of the histidine fatty aldehyde Schiff base comprises the following steps: reacting histidine with fatty aldehyde with different chain lengths in a solvent mixed with an alkaline substance at 50-90 ℃ for 3-8 hours under the protection of nitrogen to generate histidine fatty aldehyde Schiff base;
the chemical formula of the histidine fatty aldehyde Schiff base is shown as
Wherein n is any integer between 1 and 13;
further, the concentration of the histidine fatty aldehyde Schiff base is less than 2.5mmol/L.
Further, the optimal expression method is as follows: 1.5mmol/L histidine fatty aldehyde Schiff base and the activated chromobacterium violaceum are co-cultured at 30 deg.c for 24 hr, and the deoxyviolacein expressed in the thallus is collected.
The invention has the following beneficial effects:
the histidine fatty aldehyde Schiff base provided by the invention has a five-membered ring head structure and a long-chain fatty aldehyde tail part which are very similar to a signal molecule n-hexane-L-homoserine lactone of a chromobacterium violaceum quorum sensing system, and can effectively promote the expression of deoxyviolacein in chromobacterium violaceum.
Drawings
FIG. 1 histidine n-heptanal Schiff base 1 H nuclear magnetic spectrum;
FIG. 2 histidine n-octanal Schiff base 1 H nuclear magnetic spectrum;
FIG. 3 histidine-M-decanal-Schiff base 1 H nuclear magnetic spectrum;
FIG. 4 histidine dodecanal Schiff base 1 H nuclear magnetic spectrogram;
FIG. 5 deoxyviolacein mass spectrum;
FIG. 6 is a diagram of the ultraviolet full wavelength spectrum of deoxyviolacein;
FIG. 7 is a graph of a deoxyviolacein standard curve;
FIG. 8 is a sequence chart of loading of histidine fatty aldehyde Schiff base;
FIG. 9 is a graph showing 12 hours of co-culture of histidine fatty aldehyde Schiff base with Chromobacterium violaceum;
FIG. 10 effect of histidine fatty aldehyde Schiff base on the expression level of deoxyviolacein (24 hours).
Detailed Description
The present invention will be further described with reference to specific examples, but the starting materials for the present invention are commercially available unless otherwise specified.
Example 1 Synthesis of histidine fatty aldehyde Schiff base
10mmol of histidine and 10mmol of potassium hydroxide are added into a round-bottom flask containing 60mL of absolute ethanol, and the mixture is stirred and dissolved at 50 ℃ under the protection of nitrogen. 20mmol of aliphatic aldehyde was dissolved in 20mL of absolute ethanol, slowly added dropwise to a round-bottom flask, and stirred at 55 ℃ for 3 hours. After the reaction, the reaction mixture was cooled to room temperature, and unreacted histidine was removed by suction filtration. The filtrate solution was evaporated under reduced pressure (40 ℃ C.) to give a pale yellow solid. The product was placed in a 20mL centrifuge tube, 15mL acetone was added, washed with ultrasound for 10 minutes, centrifuged at 7000rpm/min for 3 minutes, washed 10 times in total to give a pale yellow solid, which was dried in vacuo to give a pale yellow solid. The structure of the material is characterized by using nuclear magnetic hydrogen spectroscopy, and the results are shown in figures 1, 2, 3 and 4.
Example 2 culture of Chromobacterium violaceum
#802 Medium: 10g of polypeptone, 2g of yeast powder and MgSO 4 ·7H 2 Adding deionized water into the O1 g to be constant volume to 1 liter, and sterilizing the mixture for 30min at 120 ℃.
In the clean bench, a sterile 24-well plate was taken. 1980. Mu.L of #802 liquid medium and 20. Mu.L of activated 12-hour chromobacterium violaceum solution were added to each well, and the mixture was cultured at 30 ℃ for 36 hours.
Example 3 identification of deoxyviolacein
The culture solution of example 1 was transferred to a centrifuge tube, centrifuged for 20 minutes (10000 r/min) to remove the upper layer, added with deionized water and ultrasonically washed for 10 minutes, and centrifuged for 20 minutes (10000 r/min) to remove the upper layer. 1mL of methanol was added to the centrifuge tube for extraction, and the mixture was centrifuged for 20 minutes (10000 r/min) to collect the supernatant, which was evaporated to dryness on a rotary evaporator. 1mL of methanol was added again for extraction and spin-dried, and the operation was repeated three times.
The extracted blue-violet pigment is deoxyviolacein with a molecular weight of 326 (figure 5) identified by mass spectrometry.
The extracted deoxyviolacein is prepared into a methanol solution with the concentration of 1mg/mL, and ultraviolet full-wavelength scanning is carried out by using a cuvette with the specification of 200 mu L, so that the characteristic absorption peak is determined to be 564.5nm (figure 6).
Example 4 Deoxyaviolacein Standard Curve assay
Preparing deoxyviolacein into methanol solutions with different concentrations, and performing absorbance value (OD) by using a cuvette with specification of 200 mu L 564.5nm ) The determination is carried out by the following steps,the origin plotting software was used to plot the standard curve (FIG. 7).
Example 5 histidine fatty aldehyde promotes the expression of deoxyviolacein in Chromobacterium violaceum
Preparing a histidine n-heptanal concentration gradient solution: in a clean bench, solutions with concentration gradients of 2.5mmol/L, 2mmol/L, 1.5mmol/L, 1mmol/L, 0.5mmol/L and 0.1mmol/L are prepared by taking sterile water as a solvent and histidine-n-heptaldehyde Schiff base as a solute, and each gradient solution is 3mL. Preparing concentration gradient solution of histidine n-octanal Schiff base, histidine decanal Schiff base and histidine dodecanal Schiff base by the same method.
In the clean bench, a sterile 24-well plate was taken. 1970. Mu.L of #802 liquid medium was added to each well, and 20. Mu.L of activated 12h Chromobacterium violaceum solution was added to each well, and 10. Mu.L of histidine fatty aldehyde Schiff base was added to each well. The loading sequence of the histidine fatty aldehyde schiff base is shown in fig. 8.
The photograph of the Schiff base histidine fatty aldehyde co-cultured with Chromobacterium violaceum at 30 ℃ for 12 hours is shown in FIG. 9. The histidine fatty aldehyde Schiff base addition group obviously turns blue, and with the increase of the concentration of a sample, the length of a fatty chain in Schiff base molecules is prolonged, and the bluish purple color of the system is deepened.
Example 6 determination of the content of deoxyviolacein
After the group of samples in example 5 is cultured for another 12 hours (total culture time is 24 hours), the culture solution in each well is respectively transferred to a centrifuge tube, centrifuged for 20 minutes (10000 r/min) to remove the upper layer, then deionized water is added for ultrasonic washing for 10 minutes, and centrifuged for 20 minutes (10000 r/min) to remove the upper layer. 1mL of methanol was added to the centrifuge tube for extraction, and the mixture was centrifuged for 20 minutes (10000 r/min) to collect the supernatant, which was evaporated to dryness on a rotary evaporator.
1mL of methanol was again added for extraction and spin-dried, and the operation was repeated three times.
Dissolving the extracted deoxyviolacein in 200 μ L methanol respectively, and detecting its absorbance value (OD) with ultraviolet spectrophotometer 564.5nm ). The expression level of Chromobacterium deoviolaceum was calculated from the standard curve (FIG. 10).
The result shows that the histidine fatty aldehyde Schiff base can obviously improve the expression quantity of the deoxyviolacein of chromobacterium violaceum; the induction effect increases with the elongation of the fatty chain of the histidine fatty aldehyde schiff base.
The foregoing examples are provided for illustration and description of the invention and are not intended to limit the invention to the described examples. Furthermore, it will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that many variations and modifications may be made in accordance with the teachings of the present invention, which variations and modifications are within the scope of the present invention as claimed.
Claims (2)
1. A method for promoting the expression of deoxyviolacein in Chromobacterium violaceum, which comprises the following steps: co-culturing 0.1-2.5 mmol/L histidine fatty aldehyde Schiff base and 12-hour activated chromobacterium violaceum at 25-35 deg.c for 12-48 hr, and collecting deoxyviolacein expressed by thallus;
the preparation method of the histidine fatty aldehyde Schiff base comprises the following steps: reacting histidine with fatty aldehyde with different chain lengths in a solvent mixed with an alkaline substance at 50-90 ℃ for 3-8 hours under the protection of nitrogen to generate histidine fatty aldehyde Schiff base;
the chemical formula of the histidine fatty aldehyde Schiff base is shown as
Wherein n is any integer between 5 and 9.
2. The method of claim 1, wherein the expression of deoxyviolacein in the chromobacterium violaceum is increased by: 1.5mmol/L histidine fatty aldehyde Schiff base and the activated chromobacterium violaceum are co-cultured at 30 deg.c for 24 hr, and the deoxyviolacein expressed in the thallus is collected.
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Citations (7)
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| WO2002050299A2 (en) * | 2000-12-20 | 2002-06-27 | Stiftung Alfred-Wegener-Institut Für Polar- Und Meeresforschung | Microbiological method for the biosynthesis of natural blue-violet colorants violacein and desoxyviolacein and the utilization thereof |
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2019
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| CN101319219A (en) * | 2008-07-11 | 2008-12-10 | 清华大学 | Method for preparing deoxidized violacein and special recombinant bacterium |
| CN102099477A (en) * | 2008-07-11 | 2011-06-15 | 清华大学 | Recombinant bacteria for producing deoxyviolacein and uses thereof |
| CN101368169A (en) * | 2008-10-17 | 2009-02-18 | 清华大学 | Pseudomonas putida for preparing deoxidized violacein and uses thereof |
| WO2012061082A2 (en) * | 2010-10-25 | 2012-05-10 | Marrone Bio Innovations, Inc. | Chromobacterium bioactive compositions and metabolites |
| CN108949600A (en) * | 2018-07-11 | 2018-12-07 | 天津大学 | The missing and its application of gene, the bacterial strain for lacking the gene and its application in the yield for improving microbial secondary metabolite |
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