CN110857448B - Method for improving chlamydomonas reinhardtii biomass and carotenoid content by using DNA (deoxyribonucleic acid) methyltransferase inhibitor - Google Patents

Method for improving chlamydomonas reinhardtii biomass and carotenoid content by using DNA (deoxyribonucleic acid) methyltransferase inhibitor Download PDF

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CN110857448B
CN110857448B CN201810970732.1A CN201810970732A CN110857448B CN 110857448 B CN110857448 B CN 110857448B CN 201810970732 A CN201810970732 A CN 201810970732A CN 110857448 B CN110857448 B CN 110857448B
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biomass
chlamydomonas reinhardtii
transferase inhibitor
dna methylation
algae
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CN110857448A (en
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姜建国
梁明华
宋德幸
梁志聪
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South China University of Technology SCUT
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    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/12—Unicellular algae; Culture media therefor

Abstract

The invention discloses a method for improving chlamydomonas reinhardtii biomass and carotenoid content by using a DNA (deoxyribonucleic acid) methylation transferase inhibitor, belonging to the technical field of food science. The method comprises the steps of culturing chlamydomonas reinhardtii cells in a liquid culture medium containing a DNA methylation transferase inhibitor for a certain time, monitoring biomass every day, collecting the algae cells, and extracting pigment. The method is simple and easy to implement, good in effect and convenient to operate, and the biomass and the carotenoid content of the chlamydomonas reinhardtii can be quickly and effectively improved after the DNA methylation transferase inhibitor is added. The DNA methylation transferase inhibitor has the advantages of very small dosage, obvious effect and convenient operation.

Description

Method for improving chlamydomonas reinhardtii biomass and carotenoid content by using DNA (deoxyribonucleic acid) methyltransferase inhibitor
Technical Field
The invention belongs to the technical field of food science, and particularly relates to a method for improving chlamydomonas reinhardtii biomass and carotenoid content by using a DNA methylation transferase inhibitor.
Background
The Chlamydomonas reinhardtii has simple culture conditions, short growth period, complete whole genome sequencing, clear genetic background and easy genetic transformation, and can accurately process, modify and fold eukaryotic proteins after translation; belongs to the safety level (GRAS) microorganism, and the expression product does not contain toxic substances, thereby being convenient for purification and reducing the production cost of protein; in addition, the chlamydomonas reinhardtii is cultured without occupying precious cultivated land resources and consuming a large amount of fertilizer and fresh water resources, and is considered as a bioreactor with great development potential. The chlamydomonas reinhardtii can produce hydrogen by utilizing solar energy and water, so that the production cost is greatly reduced, and the chlamydomonas reinhardtii is considered to be a biological hydrogen production species with very high development potential; and can also develop and utilize Chlamydomonas reinhardtii to produce high added value products such as carotenoid, grease, long-chain unsaturated fatty acid and the like.
Although Chlamydomonas reinhardtii has been regarded as a bioreactor and has some applications, some aspects are yet to be further developed and improved, one aspect is that the biomass of Chlamydomonas reinhardtii which is autotrophic through simple photosynthesis is low, so that the yield of recombinant proteins is influenced, or the yield of final target products (high value-added products including carotenoids) is reduced. High density heterotrophic fermentation or mixotrophy is a means of increasing biomass, and in addition, biomass can be increased by the induction of chemicals.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for improving the biomass of chlamydomonas reinhardtii and the content of carotenoid by using a DNA methyltransferase inhibitor. The method can improve the yield of high value-added substances such as carotenoid, grease, recombinant protein, hydrogen and the like in the chlamydomonas reinhardtii, and is an effective way for improving the biomass of the chlamydomonas reinhardtii.
The purpose of the invention is realized by the following technical scheme:
the invention provides an application of a DNA methylation transferase inhibitor in improving the biomass of Chlamydomonas reinhardtii and the content of carotenoid.
A method for increasing Chlamydomonas reinhardtii biomass and carotenoid content by using DNA methylation transferase inhibitor comprises the following steps: culturing Chlamydomonas reinhardtii cells in liquid culture medium containing DNA methyltransferase inhibitor for a certain period of time, monitoring biomass every day, collecting the cells, and extracting pigment.
The DNA methylation transferase inhibitor comprises at least one of 5-azacytidine (5-azacytidine, 5AzAC for short) and 5-aza-2 '-deoxycytidine (5-aza-2' -deoxycytidine, 5 AzdC for short).
The final concentration of the DNA methylation transferase inhibitor is 10-1000 mu M.
The culture time is 4-20 days or more.
The culture conditions are that under the conditions that the illumination intensity is 500-10000 Lx, the light dark period is 6-18 h: performing shake culture at the rotation speed of 10-200 r/min at the temperature of 15-30 ℃ for 6-18 h, preferably at the illuminance of 8000Lx and the light-dark period of 14 h: culturing at 25 deg.C for 10h with shaking at 50 r/min.
The Chlamydomonas reinhardtii liquid culture medium is preferably a TAP culture medium, and comprises the following components: tris 2.42g/L, TAP-salts (containing NH)4Cl 15g/L，MgSO4·7H2O 4g/L，CaCl2·2H2O2 g/L)25mL/L, phosphate solution (containing K)2HPO4 28.2g/100mL，KH2PO414.4g/100mL)1mL/L, trace element liquid (containing Na)2EDTA·2H2O 5g/100mL，ZnSO4·7H2O 2.2g/100mL，H3BO3 1.14g/100mL， MnCl2·4H2O 0.5g/100mL，FeSO4·7H2O 0.5g/100mL，CoCl2·6H2O 0.16g/100mL， CuSO4·5H2O 0.16g/100mL，(NH4)6Mo7O24·4H2O0.11 g/100mL)1mL/L, acetic acid 1mL/L, adjusting pH to 6.95-7.0.
The method specifically comprises the following steps:
inoculating Chlamydomonas reinhardtii cells into a liquid culture medium, adding a proper amount of DNA methylation transferase inhibitor, culturing for 4-20 days or more, monitoring biomass every day, collecting the algae cells, and extracting pigment.
The culture conditions are that under the conditions that the illumination intensity is 500-10000 Lx, the light dark period is 6-18 h: performing shake culture at the rotation speed of 10-200 r/min at the temperature of 15-30 ℃ for 6-18 h, preferably at the illuminance of 8000Lx and the light-dark period of 14 h: culturing at 25 deg.C for 10h with shaking at 50 r/min.
The biomass measuring method comprises the following steps: and (3) measuring the absorbance value of the algae liquid at the wavelength of 750nm by using an ultraviolet spectrophotometer, wherein the higher the absorbance value is, the higher the biomass is. In addition, the accumulation of biomass can be determined by measuring the dry weight of the algae cells, and the algae cells are collected by centrifugation, dried in an oven at 55 ℃ for 48 hours to constant weight and directly weighed.
The method for extracting the pigment comprises the following steps: centrifuging the cultured algae solution, discarding the supernatant, mixing the algae mud with an organic solvent, leaching, centrifuging, and filtering the supernatant with a filter membrane.
The organic solvent is acetone, ethanol, methanol or acetonitrile, and the like, preferably acetone; the leaching time is 15-30 min, and the filter membrane is a 0.45-micrometer filter membrane.
The method for measuring the pigment content comprises the following steps: acetone is used as a solvent, a spectrophotometer or an enzyme-linked immunosorbent assay is used for respectively measuring the absorbance values at the wavelengths of 665.2nm, 652.4nm and 470nm, and then the following formula is used for calculation:
chlorophyll a (μ g/mL) ═ 16.72 · a665.2-9.16·A652.4
Chlorophyll b (μ g/mL) ═ 34.09. A652.4-15.28·A665.2
Total carotenoids (μ g/mL) ═ 1000. a470-1.63·Chl a-104.96·Chl b)/221
Wherein Chl a represents chlorophyll a, and Chl b represents chlorophyll b.
Compared with the prior art, the invention has the following advantages and effects:
the method is simple and easy to implement, good in effect and convenient to operate, the biomass and the carotenoid content of the chlamydomonas reinhardtii can be quickly and effectively improved after the DNA methylation transferase inhibitor is added, probably because the DNA methylation transferase inhibitor can reduce the methylation degree of the DNA, which is equivalent to demethylation, genes which are originally silenced or have low expression level can be reactivated, and the expression of the genes is beneficial to the growth of chlamydomonas reinhardtii cells and the accumulation of pigments. The DNA methylation transferase inhibitor has the advantages of very small dosage, obvious effect and convenient operation. Moreover, many foreign genes are introduced into Chlamydomonas genome to cause gene silencing, and one reason is that methylation modification may occur, and DNA methyltransferase inhibitors can demethylate to some extent, possibly activating the expression of the foreign gene.
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FIG. 1 shows the growth of Chlamydomonas reinhardtii after 5AzAC treatment.
FIG. 2 shows the biomass and carotenoid accumulation of 5AzAC treated Chlamydomonas reinhardtii; wherein, A, Biomass (OD)750As a measure); b, carotenoid.
FIG. 3 shows the biomass and carotenoid accumulation of 5Azadc treated Chlamydomonas reinhardtii; wherein, A, Biomass (OD)750As a measure); b, carotenoid.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents and the like used are, unless otherwise specified, reagents and materials obtained from commercial sources.
The biomass measuring method comprises the following steps: and (3) measuring the absorbance value of the algae liquid at the wavelength of 750nm by using an ultraviolet spectrophotometer, wherein the higher the absorbance value is, the higher the biomass is. In addition, the accumulation of biomass can be determined by measuring the dry weight of algae cells, 100mL of algae cells are collected by centrifugation, dried in an oven at 55 ℃ for 48 hours to constant weight, and then directly weighed.
The method for extracting the pigment comprises the following steps: centrifuging the cultured algae solution, discarding the supernatant, mixing the algae mud with an organic solvent, leaching, centrifuging, and filtering the supernatant with a filter membrane.
The organic solvent is acetone, ethanol, methanol or acetonitrile, and the like, preferably acetone; the leaching time is 15-30 min, and the filter membrane is a 0.45-micrometer filter membrane.
The method for measuring the pigment content comprises the following steps: acetone is used as a solvent, a spectrophotometer or an enzyme-linked immunosorbent assay is used for respectively measuring the absorbance values at the wavelengths of 665.2nm, 652.4nm and 470nm, and then the following formula is used for calculation:
chlorophyll a (μ g/mL) ═ 16.72 · a665.2-9.16·A652.4
Chlorophyll b (μ g/mL) ═ 34.09. A652.4-15.28·A665.2
Total carotenoids (μ g/mL) ═ 1000. a470-1.63·Chl a-104.96·Chl b)/221
Wherein Chl a represents chlorophyll a, and Chl b represents chlorophyll b.
The Chlamydomonas reinhardtii used in the examples was Chlamydomonas reinhardti (Chlamydomonas reinhardti) FACHB-2220, purchased from the freshwater algae seed bank of the Chinese academy of sciences.
Example 1
(1) Cultivation of Chlamydomonas reinhardtii
Firstly, preparing a Chlamydomonas reinhardtii TAP liquid culture medium, which comprises the following components: tris 2.42g/L, TAP-salts (containing NH)4Cl 15g/L，MgSO4·7H2O 4g/L，CaCl2·2H2O2 g/L)25mL/L, phosphate solution (containing K)2HPO428.2g/100mL，KH2PO414.4g/100mL)1mL/L, trace element liquid (containing Na)2EDTA·2H2O 5g/100mL，ZnSO4·7H2O 2.2g/100mL，H3BO3 1.14g/100mL， MnCl2·4H2O 0.5g/100mL，FeSO4·7H2O 0.5g/100mL，CoCl2·6H2O 0.16g/100mL， CuSO4·5H2O 0.16g/100mL，(NH4)6Mo7O24·4H2O0.11 g/100mL)1mL/L, acetic acid 1mL/L, adjusting pH to 6.95-7.0.
Preparation of 40mM 5-azacytidine (5AzAC) stock: 0.09768g of 5AzAC powder was weighed and dissolved in 10mL of deionized water.
Chlamydomonas reinhardtii cells were then inoculated into 100mL of the above liquid medium, and 5AzAC was added to final concentrations of 0 (control), 100. mu.M, and 800. mu.M, respectively (i.e., 250. mu.L and 2000. mu.L of 40mM 5AzAC stock solution, respectively), under an illumination of 8000Lx, with a light-dark cycle of 14 h: culturing for 10h (14 h under illumination and 10h without light) (culturing alternately in dark and light), wherein the temperature is 25 ℃, the rotating speed is 50r/min, and the culturing lasts for 5-9 days.
(2) Determination of biomass
The algae liquid cultured for 5-9 days is directly observed, and the color of the algae liquid after 5 days and 9 days of 5AzAC treatment is darker than that of a control group, which shows that the cell density of algae can be improved after 5AzAC treatment (figure 1).
Taking 3-4 mL of the algae solution cultured for 5-7 days, respectively, and measuring the absorbance value of the algae solution at the wavelength of 750nm by using an ultraviolet spectrophotometer to find that the biomass of the treated group (figure 2A) is higher than that of the untreated control group.
(3) Extraction and determination of pigments
Centrifuging 2mL of algae solution at 8000r/min for 5min, discarding supernatant, adding 2mL of acetone into algae mud, vortex dispersing, leaching for 20min, centrifuging at 10000r/min for 15min, collecting upper layer of acetone, transferring to new centrifuge tube, and filtering with 0.45 μm filter membrane.
Measuring the absorbance values of the acetone phase solution at 665.2nm, 652.4nm and 470nm respectively by using a microplate reader, and calculating by using the following formula:
chlorophyll a (μ g/mL) ═ 16.72 · a665.2-9.16·A652.4
Chlorophyll b (μ g/mL) ═ 34.09. A652.4-15.28·A665.2
Total carotenoids (μ g/mL) ═ 1000. a470-1.63·Chl a-104.96·Chl b)/221
Calculating the total carotenoid according to the formula, and finding that the carotenoid content (shown in figure 2B) is obviously higher than that of an untreated control group after 100 mu M and 800 mu M5Azac treated chlamydomonas reinhardtii is cultured for 5, 6 and 7 days, wherein the carotenoid content of the 100 mu M5Azac treated group is 15.3-25.2% higher than that of the control group; the carotenoid content of the 800 mu M5AzAC treatment group is 41.9-53.3% higher than that of the control group, and the treatment effect of the 800 mu M5AzAC is the best.
Example 2
(1) Cultivation of Chlamydomonas reinhardtii
First, a Chlamydomonas reinhardtii TAP liquid medium was prepared in the same manner as in example 1.
Preparation of 40mM stock of 5-aza-2' -deoxycytidine (5 Azadc): 0.09128g of 5AzadC powder was weighed and dissolved in 10mL of deionized water.
Chlamydomonas reinhardtii cells were then inoculated into 100mL of the above liquid medium, and 5AzadC was added to final concentrations of 0 (control), 100. mu.M, and 800. mu.M, respectively (i.e., 250. mu.L and 2000. mu.L of 40mM 5AzadC stock solution, respectively), under an illumination of 8000Lx, with a light-dark cycle of 14 h: culturing for 10h (14 h under illumination and 10h without light) (culturing alternately in dark and light), wherein the temperature is 25 ℃, the rotating speed is 50r/min, and the culturing lasts for 5-7 days.
(2) Determination of biomass
Taking 3-4 mL of algae solution cultured for 5-7 days, and measuring absorbance value (OD) of algae solution at 750nm wavelength with ultraviolet spectrophotometer7s0) The biomass of the treated group (fig. 3A) was found to be higher than that of the untreated control group.
(3) Extraction and determination of pigments
The method for extracting and measuring pigment from Chlamydomonas reinhardtii is the same as that in example 1, and the calculation shows that the carotenoid content (shown in figure 3B) is higher than that of the untreated control group after the Chlamydomonas reinhardtii is added with 100 mu M and 800 mu M5 Azadc and cultured for 5, 6 and 7 days, wherein the carotenoid content of the 100 mu M5 Azadc treated group is 11.2-18.9% higher than that of the control group; the carotenoid content of the 800 mu M5 AzadC treatment group is 4.4-10.0% higher than that of the control group, and the treatment effect of 100 mu M5 AzadC is slightly better than that of 800 mu M5 AzadC. Combining example 1 and example 2, the effect of the 5AzAC treatment was better than that of the 5AzAC treatment.
Example 3
(1) Cultivation of Chlamydomonas reinhardtii
The same as example 1, but the cultivation time was 14 days.
(2) Determination of biomass
After 90mL of algal cells were collected by centrifugation, oven-dried at 55 ℃ for 48 hours to a constant weight, and directly weighed, it was found that the dry weight of algal cells in 100. mu.M and 800. mu.M 5 AzAC-treated groups was 15.2% and 17.6% higher than that in the untreated control group, respectively.
(3) Extraction and determination of pigments
The procedure for the extraction and determination of pigments from Chlamydomonas reinhardtii was the same as in example 1, and it was found by calculation that the carotenoid content was 11.4% and 10.5% higher, respectively, after Chlamydomonas reinhardtii was cultured for 14 days with the addition of 100. mu.M and 800. mu.M 5AzAC treatments.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (9)

  1. The application of a DNA methylation transferase inhibitor in improving the biomass of Chlamydomonas reinhardtii and the content of carotenoid is characterized in that:
    the DNA methylation transferase inhibitor is at least one of 5-azacytidine and 5-aza-2' -deoxycytidine.
  2. 2. Use according to claim 1, characterized in that:
    the final concentration of the DNA methylation transferase inhibitor is 100-800 mu M.
  3. 3. A method for improving the biomass and the carotenoid content of Chlamydomonas reinhardtii by using a DNA methyltransferase inhibitor is characterized by comprising the following steps:
    culturing Chlamydomonas reinhardtii cells in liquid culture medium containing DNA methylation transferase inhibitor for a certain period of time, monitoring biomass every day, collecting the cells, and extracting pigment;
    the DNA methylation transferase inhibitor is at least one of 5-azacytidine and 5-aza-2' -deoxycytidine.
  4. 4. The method of claim 3, wherein:
    the final concentration of the DNA methylation transferase inhibitor is 100-800 mu M.
  5. 5. The method of claim 3, wherein:
    the culture time is 4-20 days and more;
    the culture conditions are that under the conditions that the illumination intensity is 500-10000 Lx, the light dark period is 6-18 h: shaking culture is carried out for 6-18 h at the temperature of 15-30 ℃ and at the rotating speed of 10-200 r/min.
  6. 6. The method according to claim 3 or 5, characterized in that:
    the culture conditions are that under the conditions that the illumination intensity is 8000Lx, the light-dark period is 14 h: culturing at 25 deg.C for 10h with shaking at 50 r/min.
  7. 7. The method according to any one of claims 3 to 5, wherein:
    the liquid culture medium is TAP culture medium, and comprises the following components: 2.42g/L of Tris, 25mL/L of TAP-salts, 1mL/L of phosphate solution, 1mL/L of trace element liquid and 1mL/L of acetic acid, and adjusting the pH value to 6.95-7.0;
    the TAP-salts: NH (NH)4Cl 15g/L，MgSO4·7H2O 4g/L，CaCl2·2H2O 2g/L；
    The phosphate solution: k2HPO4 28.2g/100mL，KH2PO4 14.4g/100mL；
    The trace element liquid: na (Na)2EDTA·2H2O 5g/100mL，ZnSO4·7H2O 2.2g/100mL，H3BO3 1.14g/100mL，MnCl2·4H2O 0.5g/100mL，FeSO4·7H2O 0.5g/100mL，CoCl2·6H2O 0.16g/100mL，CuSO4·5H2O 0.16g/100mL，(NH4)6Mo7O24·4H2O 0.11g/100mL。
  8. 8. The method according to any one of claims 3 to 5, wherein:
    the biomass measuring method comprises the following steps: measuring the absorbance value of the algae liquid at the wavelength of 750nm by adopting an ultraviolet spectrophotometer, wherein the higher the absorbance value is, the higher the biomass is; or determining the accumulation condition of biomass by measuring the dry weight of the algae cells, centrifuging and collecting the algae cells, drying to constant weight, and directly weighing.
  9. 9. The method according to any one of claims 3 to 5, wherein:
    the method for extracting the pigment comprises the following steps: centrifuging the cultured algae solution, discarding the supernatant, mixing the algae mud with an organic solvent, leaching, centrifuging, and filtering the supernatant with a filter membrane;
    the organic solvent is acetone, ethanol, methanol or acetonitrile; the leaching time is 15-30 min, and the filter membrane is a 0.45-micrometer filter membrane;
    the method for measuring the pigment content comprises the following steps: measuring the absorbance values at the wavelengths of 665.2nm, 652.4nm and 470nm by using a spectrophotometer or a microplate reader, and calculating by using the following formula:
    chlorophyll a (μ g/mL) ═ 16.72 · a665.2-9.16·A652.4
    Chlorophyll b (μ g/mL) ═ 34.09. A652.4-15.28·A665.2
    Total carotenoids (μ g/mL) ═ 1000. a470-1.63·Chla-104.96·Chlb)/221
    Wherein Chla represents chlorophyll a, and Chlb represents chlorophyll b.
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