CN108977371B - Cyanobacteria strain capable of being used for production of glycerol glucoside and application thereof - Google Patents

Abstract

The invention relates to the field of microbial synthesis of high value-added chemicals. In particular to a cyanobacteria strain for producing glycerol glucoside and application thereof. The strain is cyanobacteria (cyanobacteria aponinum)1B1 which is deposited in China General Microbiological Culture Collection Center (CGMCC) of the China General Microbiological Culture Collection Committee 2017-3-2, and the Collection number is CGMCC No. 13785. The screened algae strains are separated from the natural ecological environment of the salt lake of the Shanxi Yuancheng, and have stronger GG synthesis capacity and environmental adaptability compared with the model cyanobacteria synechocystis PCC 6803; the compatible substance synthesized by the strain under salt stress is only glycerol glucoside, which is beneficial to the separation of products. Therefore, the strain is very suitable for being used as a glycerol glucoside production strain and applied to industrial production.

Description

Cyanobacteria strain capable of being used for production of glycerol glucoside and application thereof

Technical Field

The invention relates to the field of microbial synthesis of high value-added chemicals. In particular to a cyanobacteria strain for producing glycerol glucoside and application thereof.

Background

Cyanobacteria (cyanobacteria) is a prokaryotic microorganism capable of plant-type oxygen-releasing photosynthesis and is an important primary producer of marine ecosystems (Hernandez-Prieto, Semeniuk et al 2014). Compared with higher plants, cyanobacteria have the advantages of high growth rate, high photosynthetic efficiency, easy genetic modification, and the like (Zhou and Li 2010). In addition, the aquatic cyanobacteria can be cultured in a culture vessel placed in a land unsuitable for cultivation or even floating in the ocean. Based on these advantages, cyanobacteria have shown great potential in the production of biofuels, bio-based chemicals and carbohydrates with broad application prospects (Gupta, Ratha et al.2013, Angermayr, Rovira et al.2015, Hays and Ducat 2015, Sarkar and Shimizu 2015).

The production of biofuels and some important chemicals by genetically engineering cyanobacteria has been a research hotspot since 2009 (Angermayr, Hellingwerf et al 2009). Cyanobacteria have been able to synthesize a variety of important biofuels and chemicals, such as hydrogen, ethylene, isoprene, ethanol, butanol, acetone, isobutyraldehyde, isobutanol, 2, 3-butanediol, 2-methyl-1-butanol, 1, 2-propanediol, fatty acids, fatty alcohols, aliphatic hydrocarbons, sucrose, lactic acid, and 3-hydroxybutyric acid, through genetic engineering (Quintana, Van der Kooy et al.2011). Although some progress has been made in the related research, showing the great potential of cyanobacterial biosynthesis, there is currently no project that can be applied commercially. The main reasons are that on one hand, the yield of various products produced by cyanobacteria is low, the production cost is high, and no economic feasibility exists; on the other hand, the cyanobacteria scale culture technology is still about to break through (Amy T.Ma 2014). This has limited the development of genetically engineered cyanobacteria to synthesize biofuel and chemical related industries. Under the circumstances, a technical route for producing high value-added chemicals by cyanobacteria is tried to be established, so that the technical barrier from laboratory strains to large-scale application is overcome, and the industrial application of the biological production of the photosynthetic cyanobacteria is promoted.

Glycerol Glucosides (GG) are a class of substances formed by the glycosidic bond of glycerol and glucose. It has moisture keeping effect, can eliminate skin tightness after cleaning face, and can be used as cosmetic additive; meanwhile, the compound is also a macromolecular stabilizer, can inhibit the growth of bacteria and fungi, and can be used for long-term storage of protein medicines and the like; in addition, GG containing active ingredients is also found in some Japanese traditional fermented foods (such as sake and flavor enhancer), has the functions of reducing blood sugar, losing weight, treating allergic respiratory diseases and the like, and can be used as a health care product. Under the condition of salt stress, many cyanobacteria can synthesize corresponding compatible substances in cells to resist the external adverse environment and maintain the intracellular and extracellular osmotic pressure balance. These cyanobacteria can be classified into three groups according to the salt tolerance and the kind of synthetic compatible substances: one group is cyanobacteria with low salt tolerance, and compatible substances synthesized by the cyanobacteria are mainly sucrose and trehalose; the other is cyanobacteria with medium salt tolerance, and the compatible substances synthesized by the cyanobacteria are mainly glycerol glucoside; the last group is cyanobacteria with high salt tolerance, and the synthetic compatible substances of the cyanobacteria are mainly betaine and derivatives thereof (FRIEDERIKE ENGELBRECHT 1999). Therefore, research on GG synthesis of cyanobacteria and application thereof to industrial production have important economic value.

By means of genetic engineering, the synechocystis PCC6803GG transporter gene ggtCD is knocked out, so that 50% of GG accumulated by cells under salt stress is secreted to the outside of the cells, and the GG yield is increased; on the basis, GG synthesis inhibition gene ggpR is knocked out, so that GG yield can be further improved (Tan, Du et al 2015). Blocking a glycogen synthesis pathway can also significantly improve the accumulation of GG under the stress of Synechococcus PCC7002 salt (Xu, Tiago Guerra et al.2012). Semi-continuous culture of GG-producing algal strains by replacing the medium has also been shown to be an effective method for improving GG production (Tan, Du et al 2015). Furthermore, salt-stressed cyanobacterial cells, when subjected to hypotonic stress, rapidly secrete intracellular compatible substances such as GG to the outside (Reed, Warr et al 1986, Tan, Du et al 2015), a property that facilitates isolation of GG products. Although both the semi-continuous culture mode and the hypotonic stress can contribute to the production of cyanobacteria GG, they are required to collect algal cells by centrifugation for many times; the centrifugal operation will bring about an increase in cost in industrial production. Further research shows that the GG-producing cyanobacteria encapsulated by agar gel before culture can also proliferate, synthesize and secrete GG products in the gel; the cell collection by centrifugation is not needed in the semi-continuous culture process, and the culture method is an ideal culture mode for GG-producing cyanobacteria (Tan, Du et al 2015). In conclusion, the research on the mode of producing the glycerol glucoside by the cyanobacteria proves the feasibility of the technical route, but the industrial application of the method is still limited by factors such as production cost, strain field environment tolerance, scale culture technology and the like. Therefore, under the condition, cyanobacteria germplasm resources are further screened and mined, cyanobacteria strains more suitable for industrial production of glycerol glucoside are searched, and the method has important significance for promoting the industrial application of cyanobacteria biosynthesis.

Disclosure of Invention

The invention aims to provide a cyanobacteria strain suitable for producing glycerol glucoside and application thereof.

In order to achieve the purpose, the invention adopts the technical scheme that:

a cyanobacteria strain for producing glycerol glucoside is cyanobacteria (cyanobacteria aponinum)1B1 which is deposited in China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCC No.13785 from 2017-3-2.

Application of a Cyanobacterium strain capable of being used for producing glycerol glucoside, wherein Cyanobacterium (Cyanobacterium aponinum)1B1 is used for producing glycerol glucoside and glycogen.

The Cyanobacterium (Cyanobacterium aponinum)1B1 can be used for producing glycogen under the condition of salt stress or used for synthesizing and accumulating in cells to produce glycerol glucoside.

A preparation method of glycerol glucoside comprises subjecting cyanobacteria (cyanobacteria aponinum)1B1 to salt stress treatment, and allowing intracellular synthesis and accumulation to prepare glycerol glucoside.

The salt stress treatment is to culture the strain in BG11 culture medium to late logarithmic phase, then add NaCl to final concentration of 0.3-0.9M, and continue culturing for 3 days.

A method for preparing glycogen comprises culturing cyanobacteria (Cyanobacterium aponinum)1B1 under illumination, and synthesizing and accumulating glycogen in cells.

The light culture is to inoculate the strain into BG11 culture medium at 100 muE/m²The light irradiation is carried out per second, and 5 percent CO is introduced₂Culturing under the condition.

Related terms

Cyanobacteria (also known as cyanobacteria) are a class of photoautotrophic prokaryotic microorganisms that are capable of utilizing solar energy to fix carbon dioxide.

The salt tolerance is the tolerance of organisms to the extracellular high-salt environment, and refers to the tolerance to sodium chloride in the invention; the growth rate is an index for measuring the growth speed of microorganisms under specific conditions; yield is a measure of the ability of a microorganism to produce a certain metabolite under certain conditions and for a certain period of time.

The invention has the advantages that:

the screened algae strains are separated from the natural ecological environment of the salt lake of the Shanxi Yuancheng, and have stronger GG synthesis capacity and environmental adaptability compared with the model cyanobacteria synechocystis PCC 6803; the compatible substance synthesized by the strain under salt stress is only glycerol glucoside, which is beneficial to the separation of products. Therefore, the strain is very suitable for being used as a glycerol glucoside production strain and applied to industrial production.

Drawings

FIG. 1 is a photograph of an algal strain 1B1 under an optical microscope (a), a transmission electron microscope (B) and scanning electron microscopes (c and d), respectively, according to an embodiment of the present invention.

FIG. 2 is a phylogenetic analysis diagram of the 16S rRNA sequence of the strain provided by the embodiment of the present invention. As is clear from the figure, the 16S rDNA sequence of algal strain 1B1, which is a strain of cyanobacter aponinum, is most similar to AM238427, and it is highly probable that algal strain 1B1 is also a strain of cyanobacter aponinum.

FIG. 3 is a graph comparing the salt tolerance of Synechocystis PCC6803(a) with that of the identified strain 1B1 (B).

FIG. 4 is a graph showing the growth profile (a) of algal strain 1B1 of the present example and control algal strain Synechocystis PCC6803 cultured in a column photoreactor (Tan, Yao et al.2011), a glycogen yield map (B), a glycerol glucoside yield map (c) and a glycerol glucoside yield map (d) under salt stress conditions. Wherein in FIG. 4a, the time of stress with 600mM naCl is indicated by the arrow; FIG. 4c and d horizontal axis shows time after salt stress.

FIG. 5 is a peak spectrum of compatible substances of Synechocystis PCC6803 and 1B1 strains in the extracellular (a and B) and intracellular (c and d) of three days of salt stress.

Detailed Description

The invention is further explained below with reference to the drawings.

Example 1: morphological identification of algal strains

1. Optical microscope observation method

1) The power supply of the optical microscope (BX51, olympus, japan) was turned on, and the condenser lens was adjusted to have a suitable field brightness.

2) Will grow to OD ₇₃₀1 drop of 1.0 (about 4 days old) algal solution was dropped on a clean slide glass, and then the slide glass was covered with a cover glass.

3) The algal cell was found under a 40-fold objective lens, then 1 drop of cedar oil was dropped on a cover glass, and the morphology of the algal cell was observed under a 100-fold objective lens, and a cell micrograph was taken (see fig. 1).

2. Scanning electron microscope sample preparation method

1) Sampling: collecting 15mL of 1B1 algal solution (OD)₇₃₀1.0-1.5) centrifuging at 4000rpm for 1min,the supernatant was removed, and 1mL of PBS phosphate buffer (8g L) with pH 7.3 was added^-1NaCl，0.24g L^-1KH₂PO₄，0.2g L^-1KCl，1.44g L^-1Na₂HPO₄) Resuspending cells, washing for three times, centrifuging at 4000rpm for 1min after each washing, and removing supernatant;

2) fixing: resuspend the cells with 1mL of 2.5% (v/v) glutaraldehyde and fix for 1.5 h; centrifuging at 4000rpm for 1min, removing glutaraldehyde, then resuspending the cells with 1mL of the same PBS phosphate buffer solution, washing for three times, standing for 10min each time, centrifuging at 4000rpm for 1min, and removing the supernatant; resuspending the cells with 1mL osmate 1% (v/v), fixing for 1h, centrifuging at 4000rpm for 1min and then removing osmate, resuspending the cells with 1mL PBS phosphate buffer solution, washing three times, standing for 10min each time, centrifuging at 4000rpm for 1min and then removing supernatant;

3) sequentially dehydrating the sample by using 1mL of ethanol aqueous solution according to the concentration gradient of 30%, 50%, 70% and 90%, wherein each step is 15min, and then dehydrating by using 1mL of 100% ethanol solution for 15min for 2 times; then placing the sample in 1mL of mixed solution of ethanol and tert-butyl alcohol 1:1 for 15 min; finally, placing the sample in 1mL of tert-butyl alcohol for 15min for 2 times; in the period, the cells do not need to be resuspended, and the dehydrated solution is taken out by a pipette after standing;

4) placing the sample in 1mL of tert-butyl alcohol for resuspending cells, and placing the sample in a freeze dryer for freeze drying; after the sample was sufficiently dried, the sample was stuck on a sample stand by a conductive tape, and the sample was observed under a cold field emission scanning electron microscope (S-4800, Hitachi technologies, Japan) after being sprayed with gold powder.

3. Transmission electron microscope sample preparation method

1) Taking materials and fixing

(1) Collecting 15mL of 1B1 algal solution (OD)₇₃₀1.0-1.5), centrifuging at 4000rpm for 1min, removing supernatant, resuspending cells in 1mL of PBS phosphate buffer (pH 7.5), washing three times, centrifuging at 4000rpm for 1min after washing, removing supernatant, then quickly putting into 1mL of pre-fixing solution 2.5% (V/V) glutaraldehyde, resuspending cells, completely immersing the taken material in the fixing solution, and standing overnight at 4 ℃;

(2) resuspending the cells with 1mL of the PBS phosphate buffer solution, washing for 3 times, standing for 30min each time, then centrifuging at 4000rpm for 1min, and removing the supernatant;

(3) putting the washed material into 1mL of post-fixing solution 1% (v/v) osmic acid, re-suspending the cells, and standing for 2 h;

(4) resuspending the cells with 1ml PBS phosphate buffer, washing 3 times for 30min each time, then centrifuging at 4000rpm for 1min, and removing the supernatant;

2) dewatering

(1) Dehydrating the washed material with 1mL of 30%, 50%, 70%, 80%, 90%, 95% (v/v) acetone aqueous solution, and standing for 15min each time;

(2) dehydrating 1mL of anhydrous acetone for 3 times, and standing for 10min each time; the whole dehydration step does not need to resuspend cells, and the dehydration solution is taken out by a pipette after standing;

3) soaking and embedding

(1) Mixing anhydrous acetone and Spurr resin (SPI-CHEM, Shanghai Italian fruit science and technology Co., Ltd.) at a volume ratio of 7:3, adding 1mL of the mixed solution into a sample (without resuspending cells), and standing at room temperature for 5 h;

(2) mixing anhydrous acetone and Spurr resin at a volume ratio of 3:7, adding 1mL of the mixture into a sample (without resuspending cells), and standing at room temperature overnight;

(3) adding 500 mu L of pure Spurr resin into the sample (without resuspending cells), and standing at room temperature for 5 h;

(4) the tissue block was embedded in a porous rubber embedding template with pure Spurr resin, the embedding plate was put into an oven and baked, polymerized and hardened at 65 ℃ for 24 hours to form an embedded block, and the embedded block was sliced and observed under a transmission electron microscope (H-7650, Hitachi Technological Co., Ltd., Japan).

From the optical micrograph (fig. 1a), it can be seen that some of the 1B1 cells were blue-green in color and some were pale in color; the cell size is about 1-2 μm, and is irregular spherical or cylindrical. From some dividing cells, it is known that this strain performs binary division as most prokaryotic microorganisms do. The transmission electron micrograph (FIG. 1b) shows that the intracellular thylakoid membranes are irregularly arranged and have some white insoluble substances similar to polyphosphoric acid; the cell surface is densely covered with ciliated structures. Scanning electron micrographs (FIGS. 1c and d) show that the algal strain has a mucous sheath outside the cell, which may be responsible for the aggregation of the cells during growth. The morphology of the strain 1B1 is very similar to that reported in the literature for the strain Cyanobacterium aponinum (Moro, Rascio et al 2007).

Example 2: identification of algal strain species based on 16S ribosomal DNA sequence

1.1 amplification and sequencing of 16S ribosomal rDNA sequence of algal Strain B1

1mL of 1B1 algal cell culture (OD)₇₃₀1.0), centrifuging and collecting cells; resuspend with 20. mu.L sterile water and freeze-thaw repeatedly 6 times at 65 ℃ in liquid nitrogen. Centrifuge at 8000rpm for 1 minute. mu.L of the supernatant was used as a template for PCR reaction, and the 16S rDNA fragment was PCR-amplified using sgF/sgR as a primer set (PCR reaction components and procedures are shown in tables 1 and 2, respectively). The obtained PCR fragment is directly sent to a sequencing company for determination to obtain a DNA sequence shown in a sequence table 1.

TABLE 1 PCR reaction System

TABLE 2 PCR reaction procedure

Primers used in Table 3

2.1 phylogenetic analysis of the B1 algal Strain

Genebank (using blastn program (Altschul 1990) (R))https:// blast.ncbi.nlm.nih.gov/Blast.cgi) The sequence of the 16S rRNA obtained by the alignment determination in the step (A) is found to have 100% of consistency with the sequence of the 16S rRNA of a strain of Cyanobacterium aponinum PCC 10605. The 16S rRNA sequences of other representative cyanobacteria listed in Table 4 were downloaded from Genebank (Moro, Rascio et al 2007) and passed through the ClustalX program (Larkin, B) together with the 16S rRNA sequence of the 1B1 algal strainlackshields et al 2007) and the resulting alignment file was used to map the evolutionary tree (Saitou and Nei 1987, Kumar 2004) using the Neighbor joining algorithm (Neighbor joiningalgorithm) in MEGA6 software, as shown in FIG. 2.

The phylogenetic analysis result further confirms that the 1B1 strain is the strain of the Cyanobacterium aponinum, so the strain is named the Cyanobacterium aponinum 1B 1.

TABLE 4 blue algae strains for phylogenetic analysis and their 16S rRNA accession numbers

Sequence listing

SEQ ID NO:1

1B 116S DNA sequence:

ACGGGCTCTTCGGAGCTAGTGGCGGACGGGTGAGGAACGCGTGAGAACCTGCCTCAAGGTCGGGGACAACAGTTGGAAACGACTGCTAATACCGGATGAGCCGAATAGGTAAAAGATTTATCGCCTAGAGAGGGGCTCGCGTCTGATTAGCTAGATGGTGAGGTAAAGGCTTACCATGGCGACGATCAGTAGCTGGTCTGAGAGGATGAGCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTCCGCAATGGGCGAAAGCCTGACGGAGCAATACCGCGTGAGGGAGGAAGGCTCTTGGGTTGTAAACCTCAAAACTTAGGGAAGAAAAAAATGACGGTACCTAATGTAAGCATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATGCAAGCGTTATCCGGAATCATTGGGCGTAAAGAGTCCGTAGGTGGCACTTCAAGTCTGCTTTCAAAGACCGAAGCTCAACTTCGGAAAGGGAGTGGAAACTGAAGAGCTAGAGTATAGTAGGGGTAGAGGGAATTCCTAGTGTAGCGGTGAAATGCGTAGAGATTAGGAAGAACACCAGTGGCGAAGGCGCTCTACTGGGCATATACTGACACTGAGGGACGAAAGCTAGGGGAGCGAAAGGGATTAGATACCCCTGTAGTCCTAGCGGTAAACGATGGATACTAGGCGTAGTGCTGTTAGAAGGACTGTGCCGAAGCTAACGCGTTAAGTATCCCGCCTGGGGAGTACGCACGCAAGTGTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCAAGGCTTGACATCCTGCGAATCTTGGAGAAATCTGAGAGTGCCTAAGGGAACGCAGAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTCCTTAGTTGCCAGCATTAAGTTGGGGACTCTAGGGAGACCGCCGGGGAGAACTCGGAGGAAGGTGGGGATGACGTCAAGTCAGCATGCCCCTTACGTCTTGGGCTACACACGTACTACAATGGTTGGGACAAAGGGGAGCGAAACCGCGAGGTGGAGCGAATCTCATCAAACCCAGCCACAGTTCAGATTGCAGGCTGAAACTCGCCTGCATGAAGGAGGAATCGCTAGTAATCGCAGGTCAGCATACTGCGGTGAATCCGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGAAGTTGGTCACGCCCGAAGTCGTTATTCTAACCCAAGTGGAAGGAGACGCCGAAGGTGGGACTAGTGACTGGGGTGAAG

(a) sequence characteristics:

● length: 1373 base pair

● type: ribonucleic acid (DNA)

● chain type: single strand

● topology: linearity

(b) Molecular type: DNA

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: 1B1

Example 3: evaluation of salt tolerance, growth rate, and Synthesis ability of compatible substance of 1B1 algal Strain

1. Evaluation of salt tolerance of algal strains

1B1 and Synechocystis PCC6803 were inoculated into BG11 medium containing 0mM, 300mM, 600mM, and 900mM sodium chloride, respectively, at 30 ℃ and 30. mu.E/m²The algal strains were observed for growth by culturing on a shaker at 150rpm for one week with a light intensity/s. Wherein the BG11 culture medium component is 1.5g L^-1NaNO₃,40mg L^-1K₂HPO₄·3H₂O,36mg L^-1CaCl₂·2H₂O,6mg L^-1Citric acid, 6mg L^-1Ammonium ferric citrate, 1mg L^-1EDTA disodium salt, 20mg L^-1NaCO₃,2.9mg L^-1H₃BO₃,1.8mg L^{- 1}MnCl₂·4H₂O,0.22mg L^-1ZnSO₄·7H₂O,0.39mg L^-1NaMoO₄·2H₂O,0.079mg L^-1CuSO₄·5H₂O and 0.01mg L^-1CoCl₂·6H₂O。

2. Evaluation of growth Rate of algal Strain

Inoculating 1B1 strain and Synechocystis PCC6803 strain into 30mL BG11 culture medium at 30 deg.C and 30 μ E/m²Performing shaking table seed culture for one week at 150rpm under the light intensity/s; according to the following steps: inoculating 100 (volume ratio) of the seed into 400mL of fresh BG11 culture medium, and culturing at 30 deg.C in air to OD₇₃₀Reaching about 1.0; the algal solution was divided into 3 portions, 120mL of each was transferred to a column photoreactor (Tan, Yao et al.2011) and charged with 5% CO₂、30℃、30μE/m²Culturing under the condition of light intensity/s until the stage approaches, and measuring OD of algal strains 0d, 2d and 3d₇₃₀According to a formula of time-of-flight calculation

g＝(t₂-t₁)/[(lgOD_t2-lgOD_t1)/lg2]

And calculating the maximum growth rate of the algal strains.

3. Treatment and measurement of algal glycogen

1) Glycogen treatment: taking 2mL of the algae liquid when the salt stress is 0d and 4d in the salt tolerance evaluation, washing the algae liquid for 3 times by using ultrapure water, suspending the algae liquid in 400 mu L of 30% (w/v) potassium hydroxide solution, incubating the algae liquid for 2h at the temperature of 95 ℃, and then adding 1.2mL of cold absolute ethyl alcohol to the algae liquid for staying overnight at the temperature of minus 20 ℃; centrifuging at 13000rpm for 15min at 4 deg.C, removing supernatant, and washing glycogen precipitate with 70% (v/v) ethanol and anhydrous ethanol respectively twice; drying at 60 ℃ for 20min with a vacuum Concentrator (Concentrator Plus, eppendorf, germany) and dissolving the precipitate with 500 μ L of 100mM sodium acetate solution (pH 4.5); adding 20 μ L saccharifying enzyme (Amylase AG 300L, Novoxin, Denmark) diluted by 10 times to 1000 times, and saccharifying at 60 deg.C for 2 hr.

2) Glycogen assay: the glycogen was measured by diluting the treated saccharified solution 0 to 10 times using a glucose analyzer (SBA-40C, domestic) according to the established experience, and then, the saccharified solution was calibrated with a glucose standard of 10mg/mL, and 25. mu.L of the sample was used for each measurement.

4. Yield determination of algal strain glycerol glucoside

1) Salt stress culture of the strain. Inoculating 1B1 strain and Synechocystis PCC6803 strain in a columnar photoreactor containing BG11 medium, introducing 5% CO₂、30℃、30μE/m²Culturing under the condition of light intensity/s until the culture is nearly flatA stage; adding sodium chloride to the culture medium, carrying out stress culture at the final concentration of 600mM, and sampling 1mL of the sodium chloride at 0d, 1d, 2d and 3d after adding salt for determining the content of the glycerol glucoside.

2) Preparation of glycerol glucoside samples. 1mL of the algal solution sample was centrifuged at 8000rpm for 5min, and the pellet was separated from the supernatant. Diluting the supernatant with ultrapure water by a certain multiple, and filtering the diluted supernatant into an ion chromatography sample bottle by a filter. 1mL of 80% ethanol was added to the cell pellet, mixed well, and bathed in water at 65 ℃ for 4 h. Centrifuging at 8000rpm for 5min, discarding precipitate, and collecting supernatant at 55 deg.C with N₂Blow drying, adding appropriate ddH₂After dilution with O, the solution was filtered through a filter into an ion chromatography vial.

3) And (4) determination of a glycerol glucoside sample. Determination of Glycerol glucoside by ion chromatography (ICS-5000, thermo, USA); and (3) analyzing the column: dionex CarboPac^TM-PA10(4 x 250mm, Product No. 046110); the mobile phase was 200mM NaOH solution at a flow rate of 1 mL/min.

As can be seen from FIG. 3, the highest salt tolerance of strain (a) of the strain Cyanobacterium aponinum 1B1 was 900mM, but its growth was inhibited at a concentration of 600mM and the cells were aggregated; the Synechocystis PCC6803(b) can still grow under the NaCl concentration of 600mM and 900mM, but the phenomena of slow growth and yellow color appear under the concentration of 900 mM. Therefore, the highest salt tolerance of the strain 1B1 is lower than that of Synechocystis PCC 6803.

According to the calculation formula for growth generation, under the condition of non-salt stress, the growth generation time of the 1B1 algal strain is 1.122, and the growth generation time of the synechocystis PCC6803 is 2.997, and the growth rate of the algal strain 1B1 is lower than that of the synechocystis PCC6803 (FIG. 4 a). However, on the fourth day of salt stress, the glycogen production of algal strain 1B1 was 153.555mg/L, whereas that of Synechocystis PCC6803 was 116.444mg/L (FIG. 4B), indicating that the glycogen production of algal strain 1B1 was higher than that of Synechocystis PCC 6803.

Consistent with literature reports (dessplats, Folco et al 2005), synechocystis PCC6803 accumulates sucrose intracellularly early in salt stress, while intracellular GG levels peak after 2 days and remain at this level at all times; and their extracellular glycerol content was gradually increased (fig. 5 a). Sucrose synthesis was not observed in the strain 1B1, and the glycerol content was almost zero. GG content of 1B1 strain reached 164.979mg/L, 1.5 times higher than Synechocystis PCC6803 at day 3 after salt stress (FIG. 4 c). Furthermore, the biomass of 1B1 algal strain was low, so the yield of GG per cell reached 39.164mg/L/OD, which was 2.9 times that of Synechocystis PCC6803 (FIG. 4 d). Therefore, the strain 1B1 has a higher GG synthesis ability than Synechocystis PCC 6803.

Biological material sample preservation information

Bacterial strains	Accession number	Storage time
			Cyanobacterium aponinum 1B1	13785	2017-3-2

The strains are all preserved in China General Microbiological Culture Collection Center (CGMCC); address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.

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SEQUENCE LISTING

<110> institute of bioenergy and Process in Qingdao, China academy of sciences

<120> cyanobacteria strain capable of being used for glycerol glucoside production and application thereof

<130>

<160> 1

<170> PatentIn version 3.1

<210> 1

<211> 1373

<212> DNA

<213> Cyanobacterium aponinum 1B1

<220>

<221> gene

<222> (1)..(1373)

<223>

<400> 1

acgggctctt cggagctagt ggcggacggg tgaggaacgc gtgagaacct gcctcaaggt 60

cggggacaac agttggaaac gactgctaat accggatgag ccgaataggt aaaagattta 120

tcgcctagag aggggctcgc gtctgattag ctagatggtg aggtaaaggc ttaccatggc 180

gacgatcagt agctggtctg agaggatgag cagccacact gggactgaga cacggcccag 240

actcctacgg gaggcagcag tggggaattt tccgcaatgg gcgaaagcct gacggagcaa 300

taccgcgtga gggaggaagg ctcttgggtt gtaaacctca aaacttaggg aagaaaaaaa 360

tgacggtacc taatgtaagc atcggctaac tccgtgccag cagccgcggt aatacggagg 420

atgcaagcgt tatccggaat cattgggcgt aaagagtccg taggtggcac ttcaagtctg 480

ctttcaaaga ccgaagctca acttcggaaa gggagtggaa actgaagagc tagagtatag 540

taggggtaga gggaattcct agtgtagcgg tgaaatgcgt agagattagg aagaacacca 600

gtggcgaagg cgctctactg ggcatatact gacactgagg gacgaaagct aggggagcga 660

aagggattag atacccctgt agtcctagcg gtaaacgatg gatactaggc gtagtgctgt 720

tagaaggact gtgccgaagc taacgcgtta agtatcccgc ctggggagta cgcacgcaag 780

tgtgaaactc aaaggaattg acggggaccc gcacaagcgg tggagtatgt ggtttaattc 840

gatgcaacgc gaagaacctt accaaggctt gacatcctgc gaatcttgga gaaatctgag 900

agtgcctaag ggaacgcaga gacaggtggt gcatggctgt cgtcagctcg tgtcgtgaga 960

tgttgggtta agtcccgcaa cgagcgcaac cctcgtcctt agttgccagc attaagttgg 1020

ggactctagg gagaccgccg gggagaactc ggaggaaggt ggggatgacg tcaagtcagc 1080

atgcccctta cgtcttgggc tacacacgta ctacaatggt tgggacaaag gggagcgaaa 1140

ccgcgaggtg gagcgaatct catcaaaccc agccacagtt cagattgcag gctgaaactc 1200

gcctgcatga aggaggaatc gctagtaatc gcaggtcagc atactgcggt gaatccgttc 1260

ccgggtcttg tacacaccgc ccgtcacacc atggaagttg gtcacgcccg aagtcgttat 1320

tctaacccaa gtggaaggag acgccgaagg tgggactagt gactggggtg aag 1373

Claims (7)

1. A cyanobacterium strain useful for glycerol glucoside production, characterized by: the strain is cyanobacteria (Cyanobacterium aponinum)1B1, which is deposited in China General Microbiological Culture Collection Center (CGMCC) at 2017-3-2 with the preservation number of CGMCC No. 13785.

2. Use of a cyanobacterium strain useful for the production of glycerol glucosides according to claim 1, characterized in that: the cyanobacteria (a)Cyanobacterium aponinum)1B1 liveApplication in producing glycerol glucoside and glycogen.

3. Use of a cyanobacterium strain useful for the production of glycerol glucosides according to claim 2, characterized in that: the cyanobacteria (a)Cyanobacterium aponinum)1B1 in the salt stress condition to produce glycogen or in the intracellular synthesis and accumulation to produce glycerol glucoside.

4. A preparation method of glycerol glucoside is characterized in that: combining the cyanobacterium of claim 1(b)Cyanobacterium aponinum)1B1 is subjected to salt stress treatment, so that it is synthesized and accumulated in cells to prepare the glycerol glucoside.

5. A process for the preparation of glycerol glucoside according to claim 4, characterized in that: the salt stress treatment is to culture the strain in BG11 culture medium to late logarithmic phase, then add NaCl to its final concentration of 0.3-0.9M, and continue culturing for 3 days.

6. A method for producing glycogen, characterized in that: combining the cyanobacterium of claim 1(b)Cyanobacterium aponinum)1B1 is cultured under illumination, and glycogen is synthesized and accumulated in cells.

7. A method for producing glycogen according to claim 6, characterized in that: the light culture is to inoculate the strain into BG11 culture medium at 100 muE/m²The light irradiation is carried out per second, and 5 percent CO is introduced₂Culturing under the condition.

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