CN108977371A - It can be used for cyanobacteria strains and its application of glycosylglycerol production - Google Patents

It can be used for cyanobacteria strains and its application of glycosylglycerol production Download PDF

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CN108977371A
CN108977371A CN201710564602.3A CN201710564602A CN108977371A CN 108977371 A CN108977371 A CN 108977371A CN 201710564602 A CN201710564602 A CN 201710564602A CN 108977371 A CN108977371 A CN 108977371A
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cyanobacteria
glycosylglycerol
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glycogen
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CN108977371B (en
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吕雪峰
钟成伟
谈晓明
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Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
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Abstract

The present invention relates to Microbe synthesis high valuable chemicals fields.Specifically there is provided a kind of cyanobacteria strains for producing glycosylglycerol and its applications.Bacterial strain is cyanobacteria (Cyanobacterium aponinum) 1B1, China Committee for Culture Collection of Microorganisms's common micro-organisms center (China General Microbiological Culture Collection Center is preserved in 2017-3-2, CGMCC), deposit number is CGMCC No.13785.The algae strain that the present invention is screened is isolated from Salt Lake In Yuncheng Region of Shanxi natural ecological environment, has stronger GG synthesis capability and adaptive capacity to environment compared with mode cyanobacteria DNC wireless；The compatible material that algae strain is synthesized in salt stress only has glycosylglycerol, and this is a kind of, is conducive to the separation of product.Therefore it is well suited as the production bacterial strain of glycosylglycerol, and is applied to industrialized production.

Description

It can be used for cyanobacteria strains and its application of glycosylglycerol production
Technical field
The present invention relates to Microbe synthesis high valuable chemicals fields.Specifically there is provided a kind of production glycerol Portugals The cyanobacteria strains of polyglycoside and its application.
Background technique
Cyanobacteria (cyanobacteria) is that one kind can be carried out plant type and put the photosynthetic prokaryotic micro-organisms of oxygen, is marine ecosystems Important primary producer (Hernandez-Prieto, Semeniuk et al.2014).Compared with higher plant, cyanobacteria The advantages (Zhou and Li 2010) such as, photosynthetic efficiency high, easy progress genetic modification fast with the speed of growth.In addition, aquatic Cyanobacteria can be cultivated with being placed on to be not suitable for tilling the land or even swim in the incubator in ocean.Based on these Advantage, cyanobacteria present its huge potentiality in terms of production bio-fuel, biological-based chemicals and glucide, have Have wide practical use (Gupta, Ratha et al.2013, Angermayr, Rovira et al.2015, Hays and Ducat 2015,Sarkar and Shimizu 2015)。
From 2009 by genetic engineering be transformed cyanobacteria produce bio-fuel and some important chemicals at For research hotspot (Angermayr, Hellingwerf et al.2009).It is transformed by genetic engineering, cyanobacteria has been able to Synthesize hydrogen, ethylene, isoprene, ethyl alcohol, butanol, acetone, isobutylaldehyde, isobutanol, 2,3- butanediol, 2-methyl-1-butene alcohol, The a variety of important bio-fuels and change such as 1,2- propylene glycol, fatty acid, fatty alcohol, aliphatic hydrocarbon, sucrose, lactic acid and 3-hydroxybutyrate Product (Quintana, Van der Kooy et al.2011).Although correlative study has been achieved for centainly being in progress, present The great potential of cyanobacteria biosynthesis, however still it is not capable of the project of commercial applications currently.Main cause is on the one hand It is that the various Product yields that cyanobacteria produces are lower, production cost is higher, not economically feasible；Another aspect cyanobacteria scale Change culture technique still to need to be broken through (Amy T.Ma 2014).Which has limited genetic engineering cyanobacteria synthesising biological fuel and chemistry The development of product related industry.In this case, it attempts to establish the technology path that cyanobacteria produces high valuable chemicals, favorably In the technology barrier got through between laboratory strains to scale application, the industrialization of photosynthetic cyanobacteria biology manufacture is pushed to answer With.
Glycosylglycerol (GG) is a kind of substance formed by glycerol and glucose by glycosidic bond combination.It, which has, protects Wet function can eliminate the tight feeling of skin after face cleaning, can be used as cosmetic additive agent use；It is also that a kind of macromolecular is steady simultaneously Determine agent, bacterial fungus can be inhibited to grow, can be used for the long-term preservation of protein drug etc.；It is eaten in addition, fermenting in some japanese traditionals It has also been found that the GG containing active constituent, has hypoglycemic, weight-reducing, treatment anaphylaxis respiratory system disease in product (such as pure mellow wine, taste increase) The functions such as disease, may be used as health care product.Under condition of salt stress, many cyanobacterias can be in the corresponding compatibility object of synthesis intracellular Matter maintains exosmosis intracellular to flatten weighing apparatus to resist extraneous inverse ring border.According to the kind of salt resistance ability and synthesis compatible material Class, these cyanobacterias can be divided into three classes: one kind is the lower cyanobacteria of salt resistance ability, and the compatible material that they are synthesized is main It is sucrose and trehalose；Another kind of is the medium cyanobacteria of salt resistance ability, and the compatible material of this kind of cyanobacteria synthesis is mainly Glycosylglycerol；Last one kind is the very high cyanobacteria of salt resistance ability, and the compatible material of this kind of cyanobacteria synthesis is mainly Glycine betaine and its derivative (FRIEDERIKE ENGELBRECHT 1999).Therefore, the GG for studying cyanobacteria is synthesized and is answered There is important economic value for industrialized production.
By genetic engineering means, DNC wireless GG transporter gene ggtCD is knocked out, cell is enabled to exist Under salt stress accumulate GG product~50% be secreted into it is extracellular, and increase GG yield；And GG synthesis is knocked out on this basis and is inhibited Gene ggpR can further increase GG yield (Tan, Du et al.2015).Glycogen synthesis approach is blocked, it also can be significant Improve the accumulation (Xu, Tiago Guerra et al.2012) of GG under Synechococcus PCC7002 salt stress.By replacing culture medium Mode, Semi-continuous cultivation produce GG algae strain, be also proved to be a kind of method (Tan, Du et for effectively improving GG yield al.2015).It, can rapidly will be intracellular compatible when by hypotonic stress in addition, the cyanobacteria cell by salt stress Property substance such as GG, is secreted into extracellular (Reed, Warr et al.1986, Tan, Du et al.2015), this characteristic is conducive to The separation of GG product.Although Semi-continuous cultivation mode and hypotonic stress can facilitate the production of cyanobacteria GG, need They repeatedly collect frustule by centrifugation；Centrifugally operated will bring the increase of cost in industrial production again.Further The study found that the production GG cyanobacteria encapsulated before culture by agaropectin, can also be proliferated, synthesis and secretion GG is produced in glue Object；Do not need that cell is collected by centrifugation during Semi-continuous cultivation yet, be it is a kind of ideal produce GG cyanobacteria training method (Tan, Du et al.2015).To sum up, at present about the production glycosylglycerol research of mode cyanobacteria demonstrate technology path can Row, but its industrial application nevertheless suffers from the factors such as production cost, bacterial strain field environment tolerance and scale evaluation technology Limitation.Therefore, in this case, cyanobacteria germ plasm resource is further screened and excavated, more suitable industrialized production is found The cyanobacteria strains of glycosylglycerol, for pushing the application of cyanobacteria Biosynthesis Industrialization to have great importance.
Summary of the invention
The object of the present invention is to provide a kind of cyanobacteria strains of suitable production glycosylglycerol and its applications.
To achieve the above object, the invention adopts a technical scheme as:
A kind of cyanobacteria strains can be used for glycosylglycerol production, bacterial strain are cyanobacteria (Cyanobacterium Aponinum) 1B1 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center in 2017-3-2 (China General Microbiological Culture Collection Center, CGMCC), deposit number is CGMCC No.13785。
A kind of application for the cyanobacteria strains can be used for glycosylglycerol production, the cyanobacteria Application of (Cyanobacterium aponinum) 1B1 in production glycosylglycerol, glycogen.
Cyanobacteria (Cyanobacterium aponinum) 1B1 produces glycogen or intracellular under condition of salt stress It synthesizes and accumulates the application in production glycosylglycerol.
A kind of preparation method of glycosylglycerol, by described cyanobacteria (Cyanobacterium aponinum) 1B1 It is handled through salt stress, make it in synthesis intracellular and accumulates and prepare glycosylglycerol.
It is described to be handled through salt stress as in BG11 culture medium culture to late log phase, it is dense to end that NaCl is then added in bacterial strain Degree be 0.3-0.9M in, and continue culture 3 days.
A kind of preparation method of glycogen trains described cyanobacteria (Cyanobacterium aponinum) 1B1 through illumination It supports, and then dynamic accumulation glycogen in the cell.
It is described through illumination cultivation be by strain inoculated into BG11 culture medium, in 100 μ E/m2/ s illumination is passed through 5%CO2Item It is cultivated under part.
Relational language
Cyanobacteria (also referred to as cyanobacteria) is a kind of photoautotrophic prokaryotic micro-organisms, can utilize solar energy, fix two Carbonoxide.
Salt resistance ability is tolerance of the biology to extracellular hypersaline environment, tolerance of the middle finger of the present invention to sodium chloride； Growth rate is the index for measuring the growth speed of microorganism under given conditions；Yield is to measure microorganism certain condition Under in specific time certain metabolite production capacity.
Advantage for present invention:
The algae strain that the present invention is screened is isolated from Salt Lake In Yuncheng Region of Shanxi natural ecological environment, with mode cyanobacteria cytoalgae PCC6803, which is compared, has stronger GG synthesis capability and adaptive capacity to environment；The compatibility object that algae strain is synthesized in salt stress Matter only has glycosylglycerol, and this is a kind of, is conducive to the separation of product.Therefore it is well suited as the production bacterium of glycosylglycerol Strain, and it is applied to industrialized production.
Detailed description of the invention
Fig. 1 is algae strain 1B1 provided in an embodiment of the present invention respectively in optical microscopy (a), transmission electron microscope (b) and scanning electricity Photo in kind under mirror (c and d).
Fig. 2 is algae strain 16S rRNA sequential system developmental analysis figure provided in an embodiment of the present invention.Algae strain 1B1 as seen from the figure 16S rDNA sequence and AM238427 it is most close, and the algae strain be cyanobacterium aponinum, so algae strain 1B1 It is probably also cyanobaterium aponinum.
Fig. 3 is DNC wireless (a) provided in an embodiment of the present invention feelings compared with identification algae strain 1B1 (b) salt resistance ability Condition figure.
Fig. 4 be the embodiment of the present invention algae strain 1B1 and control algae strain DNC wireless pillar Photoreactor (Tan, Yao et al.2011) in cultivate growth curve chart (a), the glycogen Yield mapping (b) under condition of salt stress, glycosylglycerol Yield mapping (c) and glycosylglycerol yield figure (d).Wherein in Fig. 4 a, arrow is shown plus the time of 600mM naCl stress； Fig. 4 c and d horizontal axis is the time after salt stress.
Fig. 5 is DNC wireless and 1B1 algae provided in an embodiment of the present invention strain at salt stress three days, extracellular (a and And the compatible material peak spectrogram of (c and d) intracellular b).
Specific embodiment
Explanation that the present invention will be further explained with reference to the accompanying drawings of the specification.
Embodiment 1: the Morphological Identification of algae strain
1. optical microscopy observation method
1) optical microscopy (BX51, olympus, Japan) power supply is opened, adjustment condenser is suitable to field luminance.
2) OD will be grown to730The algae solution of=1.0 (culture about 4d) takes 1 drop to close the lid after drop is on clean glass slide Slide.
3) algae strain cell is first found under 40 times of object lens, then drop 1 drips cedar oil on the cover slip, under 100 times of object lens It observes algae strain cellular morphology, shoots cell microphoto (referring to Fig. 1).
2. SEM sample preparation method
1) it samples: taking 15mL1B1 algae solution (OD730=1.0-1.5) 4000rpm centrifugation 1min, supernatant is removed, 1mL is added PBS phosphate buffer (the 8g L of pH=7.3-1NaCl, 0.24g L-1KH2PO4, 0.2g L-1KCl, 1.44g L-1Na2HPO4) Cell is resuspended, cleans three times, every time after cleaning, 4000rpm is centrifuged 1min, removes supernatant；
2) fixed: cell, fixed 1.5h is resuspended with the glutaraldehyde 1mL of 2.5% (v/v)；It is removed after 4000rpm centrifugation 1min Cell is resuspended with identical PBS phosphate buffer 1 mL later in glutaraldehyde, cleans three times, every all over standing 10min, 4000rpm Supernatant is removed after centrifugation 1min；Cell is resuspended with the osmic acid 1mL of 1% (v/v), removes osmium after fixed 1h, 4000rpm centrifugation 1min Cell is resuspended in acid, PBS phosphate buffer 1 mL, cleans three times, every all over 10min is stood, and removes supernatant after 4000rpm centrifugation 1min；
3) successively sample is dehydrated by 30%, 50%, 70%, 90% concentration gradient with the aqueous solution 1mL of ethyl alcohol, Every step 15min, later with 100% ethanol solution of 1mL be dehydrated 15min, totally 2 times；Sample is placed in ethyl alcohol and the tert-butyl alcohol 1 later: 15min in 1 mixed liquor 1mL；Most postposition sample 15min in the 1mL tert-butyl alcohol, totally 2 times；Period without cell is resuspended, stands Dehydrating solution is taken out with liquid-transfering gun afterwards；
4) sample is placed in the 1mL tert-butyl alcohol and cell is resuspended, be put into freeze drier and be freeze-dried；Sample is abundant It after drying, is sticked on sample stage by conductive tape, metal spraying powder is placed on cold field emission scanning electron microscope (S-4800, day Vertical new and high technology Co., Ltd., Japan) under observe.
3. method for preparing transmission electron microscopy
1) it draws materials and fixed
(1) 15mL1B1 algae solution (OD is taken730=1.0-1.5), 4000rpm is centrifuged 1min, removes supernatant, uses PBS phosphorus Cell is resuspended in phthalate buffer (pH=7.5) 1mL, cleans three times, and 4000rpm is centrifuged 1min after cleaning, removes supernatant, then rapidly Before investment 1mL in fixer 2.5% (V/V) glutaraldehyde, cell is resuspended, making to draw materials is totally submerged in fixer, 4 DEG C of mistakes Night；
(2) cell is resuspended with the above-mentioned PBS phosphate buffer of 1mL, cleans 3 times, stands 30min every time, then 4000rpm It is centrifuged 1min, removes supernatant；
(3) material after cleaning is put into after 1mL in fixer 1% (v/v) osmic acid, cell is resuspended, stand 2h；
(4) cell is resuspended with 1mLPBS phosphate buffer, cleans 3 times, each 30min, then 4000rpm is centrifuged 1min removes supernatant；
2) it is dehydrated
(1) material after flushing is successively used the 30% of 1mL, 50%, 70%, 80%, 90%, the acetone of 95% (v/v) Aqueous solution dehydration stands 15min every time；
(2) 1mL anhydrous propanone is dehydrated 3 times, stands 10min every time；Entire dehydration does not have to that cell is resuspended, and uses after standing Liquid-transfering gun takes out dehydration liquid；
3) it is impregnated with and embeds
(1) anhydrous propanone and Spurr resin (SPI-CHEM, Shanghai Yi Guo Science and Technology Ltd.) is mixed by 7:3 volume ratio It closes, 1mL mixed liquor is added and (is not required to that cell is resuspended) in sample, is placed at room temperature for 5h；
(2) anhydrous propanone and Spurr resin are mixed by 3:7 volume ratio, 1mL mixed liquor is added and (is not required to weight in sample Outstanding cell), left at room temperature over night；
(3) pure 500 μ L of Spurr resin is added in sample and (cell is not resuspended), is placed at room temperature for 5h；
(4) tissue block is embedded in template with pure Spurr resin embedding in expanded rubber, embedding plate is put into baking oven and is dried Roasting, polymerization hardening, for 24 hours, forms embedded block, slice is placed on transmission electron microscope (H-7650, Hitachi's new and high technology by 65 DEG C Co., Ltd., Japan) under observe.
From light micrograph (Fig. 1 a) as it can be seen that some 1B1 cells are in blue-green, some is then in pale color；Cell is big Small is approximately 1-2 μm, in irregular spherical or cylindrical.From some cells divided it is found that algae strain and most of protokaryons Microorganism equally carries out binary fission.Transmission electron microscope photo (Fig. 1 b) display, intracellular thylakoid membrane are in irregular alignment, but also There is the white insoluble matter of some similar polyphosphoric acids；Cell surface gathers pili-like structures.Stereoscan photograph (Fig. 1 c and d) is aobvious Show that algae strain extracellularly has mucus shape glue sheath, this may be the reason of cell can be assembled during the growth process.1B1 algae strain Form and Cyanobacterium aponinum algae reported in the literature strain (Moro, Rascio et al.2007) are closely similar.
Embodiment 2: the algae strain Species estimation based on 16S ribosomal dna sequence
1. the amplification and sequencing of 1B1 algae strain 16S ribosomes rDNA sequence
Take 1mL 1B1 frustule culture (OD730=1.0) cell, is collected by centrifugation；It is resuspended with 20 μ L sterile waters, and in liquid Nitrogen and 65 DEG C multigelation 6 times.8000rpm is centrifuged 1 minute.1.5 μ L supernatants are taken to be used as the template of PCR reaction, with sgF/sgR For primer pair, PCR amplification 16S rDNA segment (PCR reacted constituent and program difference are as shown in Tables 1 and 2).Obtained PCR fragment Directly sequencing company is sent to measure, obtains the DNA sequence dna as shown in sequence table 1.
1 PCR reaction system of table
2 PCR response procedures of table
3 the primer of table
2. the Phylogenetic Analysis of 1B1 algae strain
Using blastn program (Altschul 1990) Genebank (https:// blast.ncbi.nlm.nih.gov/Blast.cgi) in compare the obtained 16S rRNA sequence of measurement, as a result, it has been found that the sequence 100% is up to the 16S rRNA sequence identity of Cyanobacterium aponinum PCC10605 algae strain.From 16S rRNA sequence (Moro, the Rascio et of other listed representative cyanobacterias in table 4 is downloaded in Genebank Al.2007), pass through ClustalX program (Larkin, Blackshields et together with the 16S rRNA sequence of 1B1 algae strain Al.2007 Multiple Sequence Alignment) is carried out, what is obtained compares adjoining algorithm (the Neighbor joining in file MEGA6 software Algorithm the drafting (Saitou and Nei 1987, Kumar 2004) of chadogram) is carried out, chadogram is as shown in Fig. 2.
Phylogenetic Analysis result further confirms that 1B1 algae strain is exactly Cyanobacterium aponinum, therefore should Algae strain is named as Cyanobacterium aponinum 1B1.
Blue algae strain and its 16S rRNA searching number of the table 4 for Phylogenetic Analysis
Sequence table
SEQ ID NO:1
1B1 16S DNA sequence dna:
ACGGGCTCTTCGGAGCTAGTGGCGGACGGGTGAGGAACGCGTGAGAACCTGCCTCAAGGTCGGGGACAA CAGTTGGAAACGACTGCTAATACCGGATGAGCCGAATAGGTAAAAGATTTATCGCCTAGAGAGGGGCTCGCGTCTGA TTAGCTAGATGGTGAGGTAAAGGCTTACCATGGCGACGATCAGTAGCTGGTCTGAGAGGATGAGCAGCCACACTGGG ACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTCCGCAATGGGCGAAAGCCTGACGGAGCAA TACCGCGTGAGGGAGGAAGGCTCTTGGGTTGTAAACCTCAAAACTTAGGGAAGAAAAAAATGACGGTACCTAATGTA AGCATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATGCAAGCGTTATCCGGAATCATTGGGCGTAAAG AGTCCGTAGGTGGCACTTCAAGTCTGCTTTCAAAGACCGAAGCTCAACTTCGGAAAGGGAGTGGAAACTGAAGAGCT AGAGTATAGTAGGGGTAGAGGGAATTCCTAGTGTAGCGGTGAAATGCGTAGAGATTAGGAAGAACACCAGTGGCGAA GGCGCTCTACTGGGCATATACTGACACTGAGGGACGAAAGCTAGGGGAGCGAAAGGGATTAGATACCCCTGTAGTCC TAGCGGTAAACGATGGATACTAGGCGTAGTGCTGTTAGAAGGACTGTGCCGAAGCTAACGCGTTAAGTATCCCGCCT GGGGAGTACGCACGCAAGTGTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGTATGTGGTTTAATT CGATGCAACGCGAAGAACCTTACCAAGGCTTGACATCCTGCGAATCTTGGAGAAATCTGAGAGTGCCTAAGGGAACG CAGAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCT CGTCCTTAGTTGCCAGCATTAAGTTGGGGACTCTAGGGAGACCGCCGGGGAGAACTCGGAGGAAGGTGGGGATGACG TCAAGTCAGCATGCCCCTTACGTCTTGGGCTACACACGTACTACAATGGTTGGGACAAAGGGGAGCGAAACCGCGAG GTGGAGCGAATCTCATCAAACCCAGCCACAGTTCAGATTGCAGGCTGAAACTCGCCTGCATGAAGGAGGAATCGCTA GTAATCGCAGGTCAGCATACTGCGGTGAATCCGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGAAGTTGG TCACGCCCGAAGTCGTTATTCTAACCCAAGTGGAAGGAGACGCCGAAGGTGGGACTAGTGACTGGGGTGAAG
(a) sequence signature:
● length: 1373 base-pairs
● type: ribonucleic acid (DNA)
● chain: single-stranded
● topological structure: linear
(b) molecule type: DNA
(c) assume: no
(d) antisense: no
(e) initial source: 1B1
The evaluation of the salt resistance ability, the speed of growth, compatible material synthesis capability of embodiment 3:1B1 algae strain
1. the evaluation of algae strain salt resistance ability
The strain of 1B1 algae and the strain of DNC wireless algae are respectively connected to containing 0mM, 300mM, 600mM and 900mM sodium chloride BG11 culture medium in, 30 DEG C, 30 μ E/m2/ s light intensity is cultivated one week on the shaking table of 150rpm, the growing state of observation algae strain. Wherein BG11 medium component is 1.5g L-1NaNO3,40mg L-1K2HPO4·3H2O,36mg L-1CaCl2·2H2O,6mg L-1 Citric acid, 6mg L-1Ferric citrate, 1mg L-1EDETATE DISODIUM, 20mg L-1NaCO3,2.9mg L-1H3BO3,1.8mg L- 1MnCl2·4H2O,0.22mg L-1ZnSO4·7H2O,0.39mg L-1NaMoO4·2H2O,0.079mg L-1CuSO4·5H2O and 0.01mg L-1CoCl2·6H2O。
2. the evaluation of algae strain growth rate
The strain of 1B1 algae and the strain of DNC wireless algae are respectively connected in 30mL BG11 culture medium, 30 DEG C, 30 μ E/m2/s Light intensity, 150rpm shaking table seed culture one week；It transfers by the inoculative proportion of 1:100 (volume ratio) into the fresh BG11 culture of 400mL In base, 30 DEG C of blowing air cultures to OD730Reach 1.0 or so；Algae solution is divided into 3 parts, every part of 120mL is transferred to pillar light reaction In device (Tan, Yao et al.2011), in logical 5%CO2、30℃、30μE/m2Culture is put down to close under the condition of culture of/s light intensity During which the platform phase measures the OD of algae strain 0d, 2d and 3d730, according to for when calculation formula
G=(t2-t1)/[(lgODt2-lgODt1)/lg2]
Calculate the maximum growth rate of algae strain.
3. the processing and measurement of algae strain glycogen
1) glycogen is handled: algae solution 2mL when salt stress 0d and 4d in above-mentioned salt resistance ability evaluation is taken, with milli-Q water 3 It is secondary, it is resuspended in the potassium hydroxide solution of 400 μ L 30% (w/v), then cold dehydrated alcohol is added in 95 DEG C of incubation 2h 1.2mL, -20 DEG C overnight；Under the conditions of 4 DEG C, 13000rpm is centrifuged 15min, removes supernatant, and glycogen precipitates respectively with 70% (v/v) Ethyl alcohol and dehydrated alcohol respectively wash twice；60 DEG C dry with vacuum decker (Concentrator Plus, eppendorf, Germany) Dry 20min, precipitating are dissolved with 500 μ L 100mM sodium acetate solutions (pH=4.5)；It is added and is diluted to 1000 times step by step by 10 times Carbohydrase (Amylase AG 300L, Novi letter, Denmark) 20 μ L, 60 DEG C of saccharification 2h.
2) glycogen measures: glycogen measurement will be handled using glucose analyser (SBA-40C, domestic) according to having there is experience Saccharified liquid afterwards, which dilutes 0-10 times, to be differed, and is calibrated with the glucose standard specimen of 10mg/mL, every time measurement 25 μ L of sample.
4. the determination of yield of algae strain glycosylglycerol
1) the salt stress culture of algae strain.The strain of 1B1 algae and the strain of DNC wireless algae are seeded in cultivate containing BG11 respectively In the pillar Photoreactor of base, in logical 5%CO2、30℃、30μE/m2Culture is to close to plateau under the condition of culture of/s light intensity； Sodium chloride coercing cultivation under its final concentration of 600mM into culture medium is added, adds after salt 0d, 1d, 2d, 3d respectively to sample 1mL and is used for The measurement of glycosylglycerol content.
2) preparation of glycosylglycerol sample.By 1mL algae solution sample, 8000rpm is centrifuged 5min, by precipitating and upper sorting It opens.After supernatant dilutes certain multiple with ultrapure water, filtered with filter into ion chromatography test sample bottle.It is added to cell precipitation 80% ethyl alcohol of 1mL mixes, 65 DEG C of water-bath 4h.8000rpm is centrifuged 5min, abandons precipitating, supernatant is at 55 DEG C with N2Drying is added and closes Suitable ddH2After O dilution, it is filled into ion chromatography test sample bottle with filter.
3) measurement of glycosylglycerol sample.The measurement of glycosylglycerol with ion chromatography (ICS-5000, Thermo, the U.S.)；Analytical column: Dionex CarboPacTM- PA10 (4*250mm, Product No.046110)；Mobile phase is 200mM NaOH solution, flow velocity 1mL/min.
From figure 3, it can be seen that the highest salt resistance ability of Cyanobacterium aponinum 1B1 algae strain (a) is 900mM, but its growth has been suppressed under 600mM concentration, and the phenomenon that cell is assembled；And cytoalgae PCC6803 (b) can still be grown under the NaCl concentration of 600 and 900mM, only occur under 900mM concentration slow growth, The phenomenon that yellowish.Therefore, the highest salt resistance ability of 1B1 algae strain is lower than DNC wireless.
According to growth for when calculation formula, under non-condition of salt stress, the growth of 1B1 algae strain for when be 1.122, and collect born of the same parents The growth of algae PCC6803 for when be 2.997, it can be seen that algae strain 1B1 growth rate be lower than DNC wireless (Fig. 4 a). However, the glycogen yield of 1B1 algae strain is 153.555mg/L, and the glycogen yield of DNC wireless at salt stress the 4th day It is 116.444mg/L (Fig. 4 b), it is seen that the glycogen yield of algae strain 1B1 is higher than DNC wireless.
Unanimously with document report (Desplats, Folco et al.2005), DNC wireless can be in salt stress early stage In intracellular accumulation sucrose, and GG content intracellular reaches peak after 2 days, and is always held at this level；And its is extracellular Glycerol content gradually increases (Fig. 5 a).Had not seen Sucrose synthesis in the strain of 1B1 algae, glycerol content also almost without.In salt stress The 3rd day 1B1 algae strain GG content reaches 164.979mg/L afterwards, is 1.5 times (Fig. 4 c) of DNC wireless.And due to 1B1 Algae strain biomass is few, therefore unit cell GG yield reaches 39.164mg/L/OD, is 2.9 times of (figures of DNC wireless 4d).Therefore, compared with DNC wireless, 1B1 algae strain has higher GG synthesis capability.
Biological material specimens preservation information
Bacterial strain Deposit number The preservation time
Cyanobacteria Cyanobacterium aponinum 1B1 13785 2017-3-2
Above-mentioned bacterial strains are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (China General Microbiological Culture Collection Center, CGMCC)；Address: Chaoyang District, Beijing City north The institute 3 of occasion West Road 1.
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SEQUENCE LISTING
<110>Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences
<120>it can be used for cyanobacteria strains and its application of glycosylglycerol production
<130>
<160> 1
<170> PatentIn version 3.1
<210> 1
<211> 1373
<212> DNA
<213>cyanobacteria 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 kind of cyanobacteria strains that can be used for glycosylglycerol production, it is characterised in that: bacterial strain is cyanobacteria It is general to be preserved in China Committee for Culture Collection of Microorganisms in 2017-3-2 by (Cyanobacterium aponinum) 1B1 Logical microorganism center (China General Microbiological Culture Collection Center, CGMCC), Deposit number is CGMCC No.13785.
2. a kind of application of the cyanobacteria strains described in claim 1 that can be used for glycosylglycerol production, it is characterised in that: Application of cyanobacteria (Cyanobacterium aponinum) 1B1 in production glycosylglycerol, glycogen.
3. the application of the cyanobacteria strains as described in claim 2 that can be used for glycosylglycerol production, it is characterised in that: institute Cyanobacteria (Cyanobacterium aponinum) 1B1 is stated to produce glycogen under condition of salt stress or in synthesis intracellular and accumulate Produce the application in glycosylglycerol.
4. a kind of preparation method of glycosylglycerol, it is characterised in that: by cyanobacteria described in claim 1 (Cyanobacterium aponinum) 1B1 is handled through salt stress, is made it in synthesis intracellular and is accumulated and prepare glycerol-glucose Glycosides.
5. pressing the preparation method of claim 4 glycosylglycerol, it is characterised in that: described to be handled through salt stress as bacterial strain exists Then NaCl is added into final concentration of 0.3-0.9M to late log phase in the culture of BG11 culture medium, and continue culture 3 days.
6. a kind of preparation method of glycogen, it is characterised in that: by cyanobacteria (Cyanobacterium described in claim 1 Aponinum) 1B1 is through illumination cultivation, and then dynamic accumulation glycogen in the cell.
7. press claim 6 glycogen preparation method, it is characterised in that: it is described through illumination cultivation be by strain inoculated to BG11 train It supports in base, in 100 μ E/m2/ s illumination is passed through 5%CO2Under the conditions of cultivate.
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