Method for improving cyanobacteria harvesting efficiency based on photoinduced cell growth

Abstract

The invention relates to a method for harvesting liquid culture of cyanobacteria. The method comprises the step of inducing cultured cyanobacteria cells to increase the length of the cyanobacteria cells before harvesting. According to the method, a light-sensitive gene regulation and control system and a gene which is regulated and controlled by the light-sensitive gene regulation and control system and affects the cellular morphology are transferred into target cyanobacteria cells, and expression of genes related to the cellular morphology is induced through irradiation of green light, so that the cell length is successfully increased; therefore, the cultured cyanobacteria can be harvested more efficiently by using a filter membrane filtration mode.

Description

Technical Field

[0001] The invention belongs to the field of microalgae cultivation, and more particularly, relates to a method for improving cyanobacteria harvesting efficiency by changing cell morphology based on light induction. Background Art

[0002] Cyanobacteria have important development potential in future global energy sustainable development and human food security. However, the common centrifugation and filtration methods used to collect cells in downstream projects consume a lot of energy.

[0003] Synechococcus el ongatus PCC 7942 is a microalgae commonly used in cyanobacteria metabolic engineering. However, the algae cells of this microalgae are relatively small, which requires a higher speed and time of the centrifuge. The use of membrane filtration to collect cells is also prone to clogging the membrane pores, causing great economic losses to production.

[0004] Therefore, if the cyanobacterial cells can be adjusted to an appropriate size before harvesting, the harvesting efficiency during membrane filtration can be greatly improved and clogging of the membrane pores can be prevented. Summary of the invention

[0005] To solve the above problems, the present invention provides a method for harvesting liquid culture of cyanobacteria, comprising the step of inducing the length of cultured cyanobacterial cells to increase before harvesting.

[0006] In a specific embodiment, a light-sensitive gene regulatory system and a gene that increases cell length and whose expression is regulated by the light-sensitive gene regulatory system are introduced into the cyanobacteria.

[0007] In a specific embodiment, the gene that increases cell length is sulA, and the encoded amino acid sequence is shown in SEQ ID NO:1.

[0008] In a specific embodiment, the light-sensitive gene regulation system is a CcaS / CcaR two-component system, which comprises a ccaS gene (sequence number SEQ ID NO: 2), two ccaR genes (sequence numbers SEQ ID NO: 3 and SEQ ID NO: 4, respectively), and a PcpcG2 promoter (sequence number SEQ ID NO: 5).

[0009] In a specific embodiment, the cyanobacterium is Synechococcuse longatus PCC 7942.

[0010] In a specific embodiment, the method comprises the following steps:

[0011] S1: subjecting the cyanobacterial liquid culture to green light induction;

[0012] S2: Filter the induced cyanobacterial liquid culture through a membrane.

[0013] In a specific embodiment, in S1, the cyanobacterial liquid culture is exposed to light at a wavelength of 660 nm and a light intensity of 5 μmol·m -2 ·s -1 Red light with a wavelength of 520nm and a light intensity of 15μmo l·m -2 ·s -1 The culture was induced under mixed light consisting of green light for two days.

[0014] In a specific embodiment, the induced cyanobacterial liquid culture is filtered using a filter membrane with a pore size of 3 μm.

[0015] The present invention successfully increases the cell length by transferring a light-sensitive gene regulatory system and a sulA gene whose expression is regulated by the light-sensitive gene regulatory system into target cyanobacterial cells, and inducing the gene expression under green light, thereby achieving a more efficient way of harvesting the cultivated microalgae by using a filter membrane. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the green light-induced gene regulation system constructed in the present invention.

[0017] Figure 2 Fluorescence micrographs of algal cells at different induction times.

[0018] Figure 3 Cell micrographs (A) and length statistics (B) of different strains.

[0019] Figure 4 This is the result of membrane filtration of the strain after green light induction. DETAILED DESCRIPTION

[0020] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0021] There are many sensory systems in cyanobacteria to regulate the physiological and metabolic activities of cells. Among them, the CcaS / CcaR two-component system from Synechocystis PCC 6803 is a typical light-sensing system. This system can upregulate the expression of the phycobilisome connection protein gene cpcG2 under green light irradiation. On this basis, we added an additional homologous response regulator CcaR2 (the protein sequence encoded by ccaR2 is the same as that encoded by ccaR, but the codons are replaced with synonymous codons to prevent homologous exchange between the two due to the same codons, resulting in gene fragment deletion), and made this gene also regulated by the promoter PcpcG2 to improve the expression of the target gene regulated by it.

[0022] The light-sensitive gene regulation system is a CcaS / CcaR two-component system, which comprises a ccaS gene (sequence SEQ ID NO: 2), two ccaR genes (sequences SEQ ID NO: 3 and SEQ ID NO: 4, respectively), and a PcpcG2 promoter (sequence SEQ ID NO: 5).

[0023] 1. Plasmid Construction

[0024] The replicon of the broad host plasmid RSF1010, the spectinomycin resistance gene, and the corresponding green light CcaS / CcaR two-component system (including the promoter PcpcG2) were connected to construct a low-copy vector pGL. The 25th codon encoding tyrosine in the MobA protein ORF in pGL was mutated to a phenylalanine codon to obtain a high-copy vector pGL2. Then, the green fluorescent protein sfGFP, cell morphology regulatory factor Su lA and EFP coding sequences were cloned into the downstream of the PcpcG2 promoter in pGL and pGL2, respectively, to obtain low-copy plasmids pGL-sfGFP, pGL-Su lA, pGL-EFP and high-copy plasmids pGL2-sfGFP, pGL2-Su lA, pGL2-EFP. The DNA fragments for constructing the above plasmids were all artificially synthesized or amplified from the corresponding genomes.

[0025] 2. Strain Construction

[0026] 1) Preparation of parental strains

[0027] The target plasmid was transformed into Escherichia coli strain DH10B (pRL443 & pRL623). After obtaining positive transformants, 3 to 5 single colonies were picked and cultured in LB liquid containing chloramphenicol (Cm), ampicillin (Car) and spectinomycin (Sp) for 5 to 10 mL. The culture was incubated in a constant temperature shaker at 37°C overnight until the late logarithmic growth phase (OD 600 =0.5~0.7).

[0028] 2) Collection and mixing of parents

[0029] Collect E. coli cells, wash the cells twice with LB medium without antibiotics, and then resuspend the cells with antibiotic-free LB medium. At the same time, collect cyanobacteria PCC 7942 in the logarithmic growth phase, wash the cells twice with BG11 medium without antibiotics to remove the old medium and cell metabolites, and then resuspend the bacteria with antibiotic-free BG11. Then mix the two bacteria in equal volumes, place them in a light incubator for 4 to 6 hours, spread them on the antibiotic-free BG11 plate, and place them in a light incubator for upright culture after the bacterial solution on the plate is absorbed by the plate.

[0030] 3) Antibiotic screening

[0031] Place the plate in a light incubator and culture at 30℃ for 24h. Then take it out and add appropriate concentrations of screening antibiotics Sp / Sm to the bottom of the culture medium in a clean bench. Keep it upright for culture. When a single colony grows on the plate, transfer the single colony to a fresh plate containing the corresponding antibiotic for expansion culture, and mark the clone number. Pick at least 5 single colonies for each conjugation. After the clones on the plate form a bacterial lawn, transfer them to a liquid culture medium for expansion culture of 5 to 10mL. Perform phenotypic verification of the clones later.

[0032] The final strains include: 7942_GFP_LC (containing low copy plasmid pGL-sfGFP), 7942_Su lA_LC (containing low copy plasmid pGL-Su lA), 7942_EFP_LC (containing low copy plasmid pGL-EFP), 7942_GFP_HC (containing high copy plasmid pGL2-sfGFP), 7942_Su lA_HC (containing high copy plasmid pGL2-Su lA), 7942_EFP_HC (containing high copy plasmid pGL2-EFP).

[0033] 3. Verification and observation of green light-induced expression system

[0034] The constructed strains 7942_GFP_LC and 7942_GFP_HC containing GFP expression plasmid were subjected to light at a wavelength of 660 nm and an intensity of 15 μmol·m -2 ·s -1 The cells were pre-cultured under red light until OD750 was about 0.3, and then the red light intensity was reduced to 5 μmol·m -2 ·s -1 , and add 15 μmol·m -2 ·s -1Green light was used to induce the expression of the target gene in the strain (pre-culture with red light was to avoid CcaR activating the expression of the target gene and reduce leakage expression; a small amount of red light was added during green light induction because red light can promote cyanobacterial cells to maintain normal photosynthesis). Samples were taken 48h, 96h, and 120h after green light induction, and the expression of fluorescent protein was observed under a fluorescence microscope. The results are shown in Figure 2 As shown in the figure, after 48 hours of green light induction, the expression of GFP in the fluorescent protein GFP expression strain increased significantly, and the fluorescence intensity was significantly higher than that before induction, indicating that it was induced to express under the regulation of green light. This shows that the green light-induced expression system constructed in this study can work normally in cyanobacterium PCC 7942 cells.

[0035] White light is mixed color light, and theoretically the green light component has a certain induction effect. Correspondingly, when activated under white light and pre-cultured under red light to the late logarithmic growth stage, the low-copy GFP protein expression strain has a small amount of fluorescent protein expression, and the high-copy expression strain has more leaky expression.

[0036] 4. Effects of light-induced Su lA and EFP expression on PCC 7942 cell size

[0037] The constructed strains 7942_SulA_LC, 7942_EFP_LC, 7942_SulA_HC, and 7942_EFP_HC were activated in liquid BG11 under white light and transferred to 24-well plates when they entered the logarithmic growth phase (OD600 was about 0.1). -2 ·s -1 The red light was pre-cultured to make it enter the mid-logarithmic growth phase (OD600 was about 0.3), and then the red light intensity was reduced to 5 μmol·m -2 ·s -1 , add 20 μmol·m -2 ·s -1 Green light was used to induce the expression of the target gene. Then, the cell morphology was observed at different time periods of white light, red light pre-culture for 72 hours, and green light induction. Figure 3 A).

[0038] Cell morphology during culture Figure 3 As shown in A, the cell length is counted as Figure 3 As shown in B.

[0039] 23% of the cells in WT_7942 were between 4.0 and 6.0 μm in length, most of them were between 2.0 and 4.0 μm in length, only 2% of the cells were between 0 and 2.0 μm, and the average length of the cells was 3.4 μm. When cultured under white light, the cell length distribution of low-copy plasmid expression strains 7942_Su lA_LC and 7942_EFP_LC had no significant difference from WT_7942, and the average cell length was 3.5μm. About 1 / 2 of the cell lengths of high-copy plasmid expression strains 7942_Su lA_HC and 7942_EFP_HC were distributed between 4.0 and 6.0μm, while 12% of the cell lengths of 7942_Su lA_HC were distributed between 6.0 and 8.0μm, and even 5% of the cell lengths were greater than 8.0μm. 12% of the cell lengths of 7942_EFP_HC were distributed between 6.0 and 8.0μm, but no cell length was greater than 8.0μm. The average lengths of strains 7942_Su lA_HC and 7942_EFP_HC were 4.7μm and 4.2μm, respectively, which were significantly greater than the average lengths of WT_7942 and low-copy plasmid expression strains.

[0040] Under white light conditions, the leaky expression of the green light-induced system caused the length of some cells of the Su lA or EFP strains to be significantly longer than that of wild-type cells.

[0041] After 72h of red light pre-culture, the cell lengths of the strains were reduced compared with those under white light. After red light pre-culture, there were almost no cells with cell lengths between 4.0 and 6.0 μm in WT_7942, about 1 / 3 of the cells were between 0 and 2.0 μm, and the rest of the cells were almost all between 2.0 and 4.0 μm, with the average cell length decreasing from 3.4 μm to 2.3 μm. After red light pre-culture, more than 80% of the cells of the low-copy plasmid expression strains 7942_Su lA_LC and 7942_EFP_LC were distributed between 2.0 and 4.0 μm, while the number of cells between 4.0 and 6.0 μm was reduced compared with that under white light, and the average length was also reduced, with the average length of 7942_Su lA_LC decreasing from 3.5 μm to 3.2 μm, and that of 7942_EFP_LC decreasing from 3.5 μm to 2.8 μm. After pre-cultured with red light, the average length of the high-copy plasmid expression strain 7942_EFP_HC strain decreased. Although the average length of the 7942_Su lA_HC strain did not change, the number of cells between 6.0 and 8.0 μm decreased compared with that under white light, and there were no cells longer than 8.0 μm.

[0042] After green light induction, the cell length of WT_7942 did not change significantly compared with the cells pre-cultured with red light, and both became shorter than when cultured under white light. After 48h and 96h of green light induction, the cell length of strains expressing EFP and Su lA did not increase significantly compared with that without induction, but the cell length increased after 120h of induction. The cell length of strain 7942_Su lA_LC was mainly distributed between 2.0 and 4.0 μm without green light induction, while the proportion of cell length between 4.0 and 6.0 μm after green light induction increased to 54% compared with 13% after red light pre-culture, and the average length also increased to 4.1 μm, showing a significant induction effect. After green light induction, the cell length of strain 7942_EFP_LC also increased compared with that after red light pre-culture, and the proportion of cell length between 4.0 and 6.0 μm increased from 6% to 21%, but there was no significant difference compared with the cell length when cultured under white light. The average cell length of the high-copy plasmid expression strain 7942_Su lA_HC did not change much after green light induction, but the number of cells with a cell length distribution of 6.0-8.0 μm or greater than 8.0 μm increased significantly. The cell length of 7942_EFP_HC also increased after green light induction compared with that after red light pre-culture, but was smaller than the average cell length under white light culture.

[0043] Based on the above results, it can be seen that the cell division inhibitor protein Su lA has a stronger effect on increasing the cell length of cyanobacterium PCC 7942 than the translation elongation factor EFP. The expression strain containing a high-copy plasmid can increase the cell length, but at the same time its leakage expression is also higher.

[0044] 5. Comparison of filtration effects induced by engineered bacteria

[0045] Before harvest, the 7942_Su lA_HC strain and the wild type were exposed to light at a wavelength of 660 nm and an intensity of 5 μ mol·m -2 ·s -1 Red light with a wavelength of 520nm and a light intensity of 15μmo l·m -2 ·s -1 The cells were induced under a mixed light consisting of green light of 100 μm and 200 μm for 3 days (green light induction). Then, the same amount of cells were taken from the two cultures, adjusted to the same volume, and filtered through a 3 μm pore size filter membrane.

[0046] The results are as follows Figure 4 As shown in the figure, the cells of the Su lA green light-induced expression strain became longer after induction, making it easier to be collected by the filter membrane. Comparison of the cell concentration in the culture before and after filtration showed that the cell collection efficiency of the green light-induced culture reached 91%, while the cell collection efficiency of the uninduced culture was only 23%. In addition, further research showed that the content of the target bioactive substance in the culture was not affected.

[0047] 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for improving the harvesting efficiency of cyanobacteria liquid culture, characterized in that: The method comprises the step of inducing the cultured cyanobacterial cells to increase in length before harvesting.

2. The method according to claim 1, characterized in that The cyanobacteria are introduced with a light-sensing gene regulatory system and genes affecting cell morphology whose expression is regulated by the light-sensing gene regulatory system.

3. The method according to claim 2, characterized in that The gene affecting cell morphology is sulA, and the encoded amino acid sequence is shown in SEQ ID NO:

1.

4. The method according to claim 3, characterized in that The light-sensitive gene regulation system is a CcaS / CcaR two-component system, and its nucleotide sequence is shown in SEQ ID NO: 2-5.

5. The method according to claim 4, characterized in that The cyanobacteria is Synechococcus elongatus PCC 7942.

6. The method according to claim 5, characterized in that The following steps are involved: S1: subjecting the cyanobacterial liquid culture to green light induction; S2: Filter the induced cyanobacterial liquid culture through a membrane.

7. The method according to claim 6, characterized in that In S1, the cyanobacterial liquid culture is exposed to light at a wavelength of 660 nm and a light intensity of 5 μmol·m -2 ·s -1 The red light with wavelength of 520nm and light intensity of 15μmol·m -2 ·s -1 The culture was induced under mixed light consisting of green light for two days.

8. The method according to claim 6, characterized in that In S2, the induced cyanobacterial liquid culture is filtered using a filter membrane with a pore size of 3 μm or larger.

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