A highly efficient carbon-fixing cyanobacterial mutant strain and its preparation method and application

Abstract

The present invention belongs to the field of biological culture technology, and specifically relates to a highly efficient carbon-fixing cyanobacteria mutant strain, a preparation method and application thereof. The preparation method of the highly efficient carbon-fixing cyanobacteria mutant strain comprises the following steps: inoculating an algae solution of elongated Synechococcus PCC 7942 into a modified BG11 culture medium, introducing air with a CO2 concentration of 10-20% into the algae solution of elongated Synechococcus PCC 7942, and continuously irradiating the algae solution at a culture temperature of 30-35°C for 3-5 cycles, wherein the culture time for each cycle is 5-10 days, and at the beginning of each cycle, the algae solution concentration of elongated Synechococcus PCC 7942 is adjusted to the initial inoculation concentration, and the highly efficient carbon-fixing cyanobacteria mutant strain is obtained after the evolution is completed. The highly efficient carbon-fixing cyanobacteria mutant strain obtained by the present invention has a relatively fast growth rate and bioaccumulation amount, and can increase the content of high value-added products such as lipids and proteins while efficiently fixing carbon.

Description

Technical Field

[0001] The invention belongs to the technical field of biological cultivation, and in particular relates to a highly efficient carbon-fixing cyanobacteria mutant strain and a preparation method and application thereof. Background Art

[0002] Synechococcus elongatus PCC 7942 (Synechococcus elongatus PCC 7942, S. elongatus PCC7942 is a freshwater unicellular cyanobacterium and one of the model organisms for studying the physiological functions and morphogenesis of cyanobacteria. PCC is the abbreviation of the Pasteur Culture Collection of the Pasteur Institute in France, and 7942 is the number of Synechococcus. Synechococcus elongatus PCC 7942 belongs to the Cyanophyta, Chroococcales, Chroococcaceae, and Synechococcus. The cells are oblong or short rod-shaped, and a single or a few cells are connected front and back to form a short filament. The reproduction method is asexual, and the photosynthesis process is similar to that of higher plants, and oxygenic photosynthesis can be carried out. Synechococcus elongatus PCC 7942 is also the first cyanobacterium that has been proven to be transformed by exogenous DNA. As a model organism, it is widely used in basic research and industrial applications due to its simple structure, high photosynthetic efficiency and simple genetic background.

[0003] Synechococcus 7942 is a prokaryotic algae. It has no nucleus, but contains nuclear material in the center of the cell, which is usually granular or reticular. Chromatin and pigment are evenly distributed in the cytoplasm. Synechococcus 7942 has no chloroplasts, but contains thylakoids, which are the main site of photosynthesis. Attached to the thylakoid membrane are phycobilisomes composed of phycocyanin and allophycocyanin, auxiliary pigment protein complexes composed of chlorophyll a, carotenoids and xanthophylls, and various electron donors of the photosynthetic and respiratory electron transport chains. As a model cyanobacterium, Synechococcus 7942 has completed genome sequencing. Research on photosynthesis, material synthesis and stress resistance targeting this algae has made certain progress.

[0004] Synechococcus 7942 has many research advantages. First, it can grow photoheterotrophically. In a light environment, it can use exogenously added inorganic substances for metabolic growth. Secondly, its genome sequencing has been completed, and it has a relatively clear genetic background. In addition, it has a natural transformation system, which makes genetic manipulation and the construction of mutant strains simple. The main advantage of Synechococcus 7942 as a chassis is that it can capture energy from sunlight through photosynthesis and convert CO2 into target products, which plays a role in carbon fixation and emission reduction while producing high-value products. Compared with eukaryotic algae and plants, Synechococcus 7942 has obvious advantages. It can convert up to 9% of solar energy into biomass, which is much higher than the 0.5-3% of higher plants. The growth rate of cyanobacteria is much faster than that of higher plants. There is no differentiation of roots, stems, leaves, flowers and fruits, which can direct more carbon and energy flows to target products. And its genetic manipulation methods have been systematized, making it easy to transform metabolic pathways. In contrast, the genetic complexity of eukaryotic algae makes its metabolic engineering more challenging. Additionally, Synechococcus can be cultivated in bioreactors on arid lands, minimizing competition with crops for land.

[0005] Synechococcus 7942 has broad prospects in efficient carbon fixation and high value-added product production. Using industrial waste gas as a carbon source to cultivate algae can not only reduce the cultivation cost of microalgae, but also make great contributions to environmental governance. However, microalgae have weak tolerance to high concentrations of CO2. Generally, CO2 greater than 10% will inhibit the algae, limiting its further application in industrial waste gas. Summary of the invention

[0006] The present invention provides a highly efficient carbon-fixing cyanobacteria mutant strain and a preparation method and application thereof. The highly efficient carbon-fixing cyanobacteria mutant strain has a faster growth rate and bioaccumulation amount, and can increase the content of high value-added products such as lipids and proteins while efficiently fixing carbon.

[0007] The specific technical solutions are as follows: The first object of the present invention is to provide a method for preparing a highly efficient carbon fixation cyanobacteria mutant, comprising the following steps: inoculating a cyanobacterial liquid into a modified BG11 culture medium, introducing air with a CO2 concentration of 10-20% into the cyanobacterial liquid, and performing evolution under continuous light at a culture temperature of 30-35°C for 3-5 cycles, wherein the culture time for each cycle is 5-10 days, and at the beginning of each cycle, adjusting the concentration of the cyanobacterial liquid to an initial inoculation concentration OD 750 =0.05-0.2, and after the evolution is completed, the highly efficient carbon-fixing cyanobacteria mutant strain is obtained.

[0008] The highly efficient carbon-fixing cyanobacteria mutant obtained by the above preparation method can use CO2 as a carbon source. The highly efficient carbon-fixing cyanobacteria mutant has a fast growth rate in a high concentration CO2 environment and has a high bioaccumulation amount, carbon fixation rate, and lipid and protein accumulation.

[0009] Furthermore, the cyanobacteria is Synechococcus elongatus PCC 7942, Synechococcus PCC 7002 or Synechocystis 6803.

[0010] Furthermore, when the light is continuously irradiated, the evolution is performed for 5 cycles.

[0011] The beneficial effects of adopting the above scheme are: CO2 concentrations between 5-8% have a weaker stress on cyanobacteria, and microalgae cells can adapt to this concentration of CO2 in a short period of time through metabolic regulation; however, CO2 concentrations exceeding 8% are a stronger stress on Synechococcus, so in order to enhance the adaptability of microalgae to high concentrations of CO2, 5 cycles of evolution were carried out.

[0012] Further, the improved BG11 medium includes the following components: 1.5-2.5 g / L NaNO3, 0.04 g / L K2HPO3·3H2O, 0.075 g / L MgSO4·7H2O, 0.036 g / L CaCl2·2H2O, 0.006 g / L C6H8O7, 0.006 g / L ferric ammonium citrate, 0.001 g / L EDTA, 0.02 g / L Na2CO3, 0.00181 g / L MnCl2·H2O, 0.00286 g / L boric acid, 0.000222 g / L ZnSO4·7H2O, 0.000079 g / L CuSO4·5H2O, 0.00039 g / L Na2MoO4·2H2O, 0.000049 g / L Co(NO3)2·6H2O.

[0013] Furthermore, the pH value of the improved BG11 medium is 7.1±0.1.

[0014] Furthermore, the illumination intensity of the illumination is 100-200 μmol·m -2 ·s -1 .

[0015] Furthermore, the gas flow rate of the air is 10-40 mL / min.

[0016] Furthermore, the evolution is carried out in a bubble reactor having a volume of 500-1000 mL.

[0017] Furthermore, the amount of the cyanobacteria liquid introduced is 300-500 mL.

[0018] The second object of the present invention is to provide a highly efficient carbon-fixing cyanobacterial mutant strain obtained by the preparation method described above.

[0019] The third object of the present invention is to provide a use of the highly efficient carbon fixation cyanobacteria mutant as described above in highly efficient carbon fixation, oil and protein production.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) The highly efficient carbon-fixing cyanobacteria, Synechococcus elongatus PCC 7942 mutant obtained by the preparation method of the present invention has a higher growth rate, bioaccumulation, lipid and protein yields in a high concentration CO2 environment; compared with the existing Synechococcus 7942 strain, it has achieved a key breakthrough in the tolerance of cyanobacteria to high concentration CO2 and efficient carbon fixation; (2) The mutant strain S20 obtained by the preparation method of the present invention has a faster growth rate, carbon fixation rate, and bioaccumulation in a 20% CO2 environment, and the biomass can reach 1.37 g / L after 7 days of cultivation; (3) The mutant strain S20 obtained by the preparation method of the present invention efficiently fixes carbon in a 20% CO2 environment while increasing the content of high value-added products such as lipids. After culturing at 30°C for 7 days, the oil content can reach 54.35 mg / g; (4) The mutant strain S20 obtained by the preparation method of the present invention can efficiently fix carbon in a 20% CO2 environment while increasing the content of high value-added products such as protein. After culturing at 30°C for 7 days, the protein content can reach 32.76 mg / g; (5) The mutant strain S20 obtained by the preparation method of the present invention has high carbon fixation efficiency under a 20% CO2 environment and has a high photosynthetic electron transfer rate and a high photosynthetic carbon fixation enzyme activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a test diagram of the bioaccumulation of the Synechococcus elongatus PCC 7942 mutant of the present invention; Figure 2 This is a test graph of the carbon fixation rate of the Synechococcus elongatus PCC 7942 mutant of the present invention; Figure 3 This is a test graph of lipid accumulation of the Synechococcus elongatus PCC 7942 mutant of the present invention; Figure 4 This is a graph showing the protein accumulation of the Synechococcus elongatus PCC 7942 mutant of the present invention. DETAILED DESCRIPTION

[0022] The principles and features of the present invention are described below in conjunction with examples. The examples are only used to explain the present invention and are not used to limit the scope of the present invention.

[0023] Example 1 The invention discloses a method for preparing a mutant strain of Synechococcus elongatus PCC 7942, which is an efficient carbon-fixing cyanobacterium. The method comprises the following steps: inoculating an algae solution of Synechococcus elongatus PCC 7942 into a modified BG11 culture medium, introducing air with a CO2 concentration of 20% into the algae solution of Synechococcus elongatus PCC 7942, and performing evolution for 5 cycles under continuous illumination at a culture temperature of 30°C. The culture time for each cycle is 7 days. At the beginning of each cycle, the algae solution concentration of Synechococcus elongatus PCC 7942 is adjusted to an initial inoculation concentration OD 750 = 0.2, after the evolution, the highly efficient carbon-fixing cyanobacteria-Schnecocos elongatus PCC 7942 mutant was obtained; The improved BG11 medium includes the following components: 2.5 g / L NaNO3, 0.04 g / L K2HPO3·3H2O, 0.075 g / L MgSO4·7H2O, 0.036 g / L CaCl2·2H2O, 0.006 g / L C6H8O7, 0.006 g / L ferric citrate ammonium, 0.001 g / L EDTA, 0.02 g / L Na2CO3, 0.00181 g / L MnCl2·H2O, 0.00286 g / L boric acid, 0.000222 g / L ZnSO4·7H2O, 0.000079 g / L CuSO4·5H2O, 0.00039 g / L Na2MoO4·2H2O, 0.000049 g / L Co(NO3)2·6H2O; the pH value of the improved BG11 medium is 7; The gas flow rate of the air is 40 mL / min; The illumination intensity of the illumination was 200 μmol·m -2 ·s -1 ; The evolution is carried out in a bubble reactor, the volume of which is 1000 mL; the inlet volume of the Synechococcus elongatus PCC 7942 algae solution is 500 mL.

[0024] Example 2 The invention discloses a method for preparing a mutant strain of Synechococcus elongatus PCC 7942, which is an efficient carbon-fixing cyanobacterium. The method comprises the following steps: inoculating an algae solution of Synechococcus elongatus PCC 7942 into a modified BG11 culture medium, introducing air with a CO2 concentration of 15% into the algae solution of Synechococcus elongatus PCC 7942, and performing evolution for 5 cycles under continuous illumination at a culture temperature of 32°C. The culture time for each cycle is 10 days. At the beginning of each cycle, the algae solution concentration of Synechococcus elongatus PCC 7942 is adjusted to an initial inoculation concentration OD 750=0.15, after the evolution, the highly efficient carbon-fixing cyanobacteria-Schnecocos elongatus PCC 7942 mutant was obtained; The components of the modified BG11 medium refer to those of Example 1, and the difference from Example 1 is that 2.5 g / L NaNO3 is replaced by 2 g / L NaNO3; the pH value of the modified BG11 medium is 7.1; The gas flow rate of the air is 25 mL / min; The illumination intensity of the illumination is 150 μmol·m -2 ·s -1 ; The evolution is carried out in a bubble reactor, the volume of which is 800 mL; the inlet volume of the Synechococcus elongatus PCC 7942 algae solution is 400 mL.

[0025] Example 3 The invention discloses a method for preparing a mutant strain of Synechococcus elongatus PCC 7942, which is an efficient carbon-fixing cyanobacterium. The method comprises the following steps: inoculating an algae solution of Synechococcus elongatus PCC 7942 into a modified BG11 culture medium, introducing air with a CO2 concentration of 10% into the algae solution of Synechococcus elongatus PCC 7942, and performing evolution for 3 cycles under continuous illumination at a culture temperature of 35°C. The culture time for each cycle is 5 days. At the beginning of each cycle, the algae solution concentration of Synechococcus elongatus PCC 7942 is adjusted to an initial inoculation concentration OD 750 = 0.05, after the evolution, the highly efficient carbon-fixing cyanobacteria-Schnecocos elongatus PCC 7942 mutant was obtained; The components of the modified BG11 medium refer to those of Example 1, and the difference from Example 1 is that 2.5 g / L NaNO3 is replaced by 1.5 g / L NaNO3; the pH value of the modified BG11 medium is 7.2; The gas flow rate of the air is 10 mL / min; The illumination intensity of the illumination was 100 μmol·m -2 ·s -1 ; The evolution is carried out in a bubble reactor, the volume of which is 500 mL; the inlet volume of the Synechococcus elongatus PCC 7942 algae solution is 300 mL.

[0026] test: 1. Tests on bioaccumulation, growth rate and carbon fixation rate of Synechococcus elongatus PCC 7942 mutant: The elongated Synechococcus PCC 7942 mutant (S20) prepared in Example 1 and the wild-type Synechococcus 7942 (WT) were inoculated in a 20% CO2 environment for culture, the elongated Synechococcus PCC 7942 mutant (S15) prepared in Example 2 and the wild-type Synechococcus 7942 (WT) were inoculated in a 15% CO2 environment for culture, and the elongated Synechococcus PCC7942 mutant (S10) prepared in Example 3 and the wild-type Synechococcus 7942 (WT) were inoculated in a 10% CO2 environment for culture. The above culture processes all used modified BG11 as the culture medium, with continuous illumination and cultured at 30°C for 7 days. During the culture process, 3 mL of microalgae culture solution was taken every 24 h, and the absorbance at a wavelength of 750 nm was measured by a microplate reader. At the same time, 20 mL of algae solution was collected, 8000 g Centrifuge for 10 min. Wash with deionized water three times and freeze-dry for 48 h before collecting algae powder for mass measurement. 750 There is a linear relationship between them, so the following formula is obtained by fitting to estimate the biomass dry weight of microalgae during the entire cultivation process. Bioaccumulation (biomass dry weight, DW, g / L) = 0.0456 xOD 750 +0.0034, r 2 = 0.998. The bioaccumulation results are shown in Figure 1 .

[0027] Depend on Figure 1 It can be seen that the mutant strain S20 obtained in Example 1 of the present invention can reach a biomass dry weight of 1.37 g / L after 7 days of cultivation in a 20% CO2 environment, while the biomass dry weight of the wild-type Synechococcus 7942 is only 0.31 g / L, and the biomass dry weight of the mutant strain S20 is 3.4 times higher than that of the wild-type Synechococcus 7942. This shows that the bioaccumulation amount of the mutant strain of elongated Synechococcus PCC 7942 is significantly higher than that of the wild-type Synechococcus 7942. On the 7th day of cultivation, the bioaccumulation amount of the wild-type Synechococcus 7942 has almost no increase, indicating that the growth rate of the mutant strain is much higher than that of the wild-type Synechococcus 7942.

[0028] After culturing at 30°C for 7 days, an appropriate amount of algae liquid was centrifuged and freeze-dried. The algae powder was then taken to measure the total carbon content in the microalgae cells using an elemental analyzer (Elementar, Germany), and the carbon fixation rate of the algae strain was calculated. The results are shown in Figure 2 shown.

[0029] Depend on Figure 2It can be seen that the maximum carbon fixation rate of S20 in Example 1 under a 20% CO2 environment can reach 0.34 g / (L·d), which is 10.3 times higher than 0.03 g / (L·d) of the wild-type Synechococcus 7942; the maximum carbon fixation rate of S15 in Example 2 can reach 0.17 g / (L·d) under a 15% CO2 environment, which is 16 times higher than 0.01 g / (L·d) of the wild-type Synechococcus 7942; the maximum carbon fixation rate of S10 in Example 3 can reach 0.25 g / (L·d) under a 10% CO2 environment, which is 7.3 times higher than 0.03 g / (L·d) of the wild-type Synechococcus 7942.

[0030] 2. Test on lipid accumulation of Synechococcus elongatus PCC 7942 mutant: The elongated Synechococcus PCC 7942 mutant (S20) prepared in Example 1 and the wild-type Synechococcus 7942 (WT) were inoculated in a 20% CO2 environment for culture, the elongated Synechococcus PCC 7942 mutant (S15) prepared in Example 2 and the wild-type Synechococcus 7942 (WT) were inoculated in a 15% CO2 environment for culture, and the elongated Synechococcus PCC 7942 mutant (S10) prepared in Example 3 and the wild-type Synechococcus 7942 (WT) were inoculated in a 10% CO2 environment for culture. The above culture process used BG11 as the culture medium, continued illumination, and cultured at 30°C for 7 days. After the culture was completed, an appropriate amount of algae solution was aliquoted and added to a 1.5 mL centrifuge tube, 50 μL of 15 μg / mL Nile red was added, and the centrifuge tube was incubated in the dark at room temperature for 10 minutes. 200 μL was taken and the sample was analyzed using a multifunctional microplate reader (using Spectramax M3 multimode spectrophotometer (Molecular Devices) records fluorescence at 530 nm (excitation) and 575 nm (emission), with a cutoff wavelength of 570 nm. The oil content of the algae strain can then be calculated using the standard curve. The results are shown in Figure 2. Figure 3 shown.

[0031] Depend on Figure 3 It can be seen that after the mutant strain S20 of Example 1 was cultured in a 20% CO2 environment for 7 days, its oil content could reach 54.35 mg / g, which was 0.51 times higher than that of the wild-type Synechococcus 7942 (36.09 mg / g); after the mutant strain S15 of Example 2 was cultured in a 15% CO2 environment for 7 days, its oil content could reach 21.07 mg / g, which was 0.06 times higher than that of the wild-type Synechococcus 7942 (19.80 mg / g); after the mutant strain S10 of Example 3 was cultured in a 10% CO2 environment for 7 days, its oil content could reach 30.62 mg·g –1 , which was 0.18 times higher than that of wild-type Synechococcus 7942 (25.88 mg / g).

[0032] 3. Protein accumulation test of Synechococcus elongatus PCC 7942 mutant: The elongated Synechococcus PCC 7942 mutant (S20) prepared in Example 1 and the wild-type Synechococcus 7942 (WT) were inoculated in a 20% CO2 environment for culture, the elongated Synechococcus PCC 7942 mutant (S15) prepared in Example 2 and the wild-type Synechococcus 7942 (WT) were inoculated in a 15% CO2 environment for culture, and the elongated Synechococcus PCC 7942 mutant (S10) prepared in Example 3 and the wild-type Synechococcus 7942 (WT) were inoculated in a 10% CO2 environment for culture. In the above culture process, BG11 was used as the culture medium, and the light was continuously illuminated and cultured at 30°C for 7 days. An appropriate amount of algae liquid was centrifuged, 1 mL of 1M NaOH was added, vortexed and mixed, and then heated in a 95°C metal bath for 10 min. After alkaline lysis, 0.5 mL of 1.6M HCI was added for neutralization. After the sample was cooled to room temperature, it was centrifuged at 13400 rpm for 10 min, the protein content was measured by BCA protein method, and the protein content was calculated by the standard curve. The results are as follows Figure 4 shown.

[0033] Depend on Figure 4 It can be seen that after the mutant S20 of Example 1 was cultured in a 20% CO2 environment for 7 days, its protein content could reach 32.76 mg / g, which was higher than that of the wild type (14.48 mg·g –1 ) increased by 1.26 times; after the mutant strain S15 of Example 2 was cultured in a 15% CO2 environment for 7 days, its protein content could reach 45.03 mg / g, which was 0.23 times higher than that of the wild type (36.55 mg / g); after the mutant strain S10 of Example 3 was cultured in a 10% CO2 environment for 7 days, its protein content could reach 38.60 mg / g, which was 0.20 times higher than that of the wild type (32.10 mg / g).

[0034] In summary, the mutant strain of Synechococcus elongatus PCC 7942 prepared by the present invention can tolerate up to 20% CO2, and has a faster growth rate, a higher bioaccumulation amount and a carbon fixation rate, which proves that the mutant strain can accumulate higher lipids and proteins while fixing carbon, and also proves that cyanobacteria have the potential to treat high-concentration industrial waste gas and produce bioenergy.

[0035] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a highly efficient carbon-fixing cyanobacterial mutant, characterized in that: The method comprises the following steps: inoculating the cyanobacteria liquid into the modified BG11 medium, introducing air with a CO2 concentration of 10-20% into the cyanobacteria liquid, and performing evolution under continuous light at a culture temperature of 30-35°C for 3-5 cycles, wherein the culture time for each cycle is 5-10 days, and at the beginning of each cycle, adjusting the concentration of the cyanobacteria liquid to the initial inoculation concentration OD 750 =0.05-0.2, and after the evolution is completed, the highly efficient carbon-fixing cyanobacteria mutant strain is obtained.

2. The method for preparing a highly efficient carbon-fixing cyanobacteria mutant according to claim 1, characterized in that: The cyanobacteria are Synechococcus elongatus PCC 7942, Synechococcus PCC 7002 or Synechocystis 6803.

3. The method for preparing a highly efficient carbon-fixing cyanobacteria mutant according to claim 1, characterized in that: When the light is continuous, the evolution is performed for 5 cycles.

4. The method for preparing a highly efficient carbon-fixing cyanobacteria mutant according to claim 1, characterized in that: The improved BG11 medium comprises the following components: 1.5-2.5 g / L NaNO3, 0.04 g / L K2HPO3·3H2O, 0.075 g / L MgSO4·7H2O, 0.036 g / L CaCl2·2H2O, 0.006 g / L C6H8O7, 0.006 g / L ferric ammonium citrate, 0.001 g / L EDTA, 0.02 g / L Na2CO3, 0.00181 g / L MnCl2·H2O, 0.00286 g / L boric acid, 0.000222 g / L ZnSO4·7H2O, 0.000079 g / L CuSO4·5H2O, 0.00039 g / L Na2MoO4·2H2O, 0.000049 g / L Co(NO3)2·6H2O.

5. The method for preparing a highly efficient carbon-fixing cyanobacteria mutant according to claim 4, characterized in that: The pH value of the improved BG11 medium is 7.1±0.

1.

6. The method for preparing a highly efficient carbon-fixing cyanobacteria mutant according to claim 1, characterized in that: The illumination intensity of the illumination is 100-200 μmol·m -2 ·s -1 .

7. The method for preparing a highly efficient carbon-fixing cyanobacteria mutant according to claim 1, characterized in that: The gas flow rate of the air is 10-40 mL / min.

8. The method for preparing a highly efficient carbon-fixing cyanobacteria mutant according to claim 1, characterized in that: The evolution is carried out in a bubble reactor, the volume of the bubble reactor is 500-1000 mL, and the access volume of the cyanobacteria liquid is 300-500 mL.

9. A highly efficient carbon fixation cyanobacterial mutant, characterized in that: The method is adopted as claimed in any one of claims 1 to 8.

10. Use of the highly efficient carbon fixation cyanobacteria mutant strain as claimed in claim 9 in highly efficient carbon fixation, production of oils and proteins.

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