CN104651287B - A kind of engineering bacteria and application for synthetic glycerine glucoside - Google Patents
A kind of engineering bacteria and application for synthetic glycerine glucoside Download PDFInfo
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Abstract
This disclosure relates to Microbe synthesis high valuable chemicals field, specifically a kind of engineering bacteria and application for synthetic glycerine glucoside.Engineering bacteria synthesizes the cyanobacteria mutant of negative regulation protein gene missing for GG movement systems gene and/or GG.Under the engineering bacteria illumination solar energy can be utilized to fix carbon dioxide growth, and under salt stress effect, it can synthesize, accumulate and secrete glycosylglycerol.The construct of synthetic glycerine glucoside of the present invention, includes the carrier of the construct, the cyanobacteria converted comprising the construct or with the carrier, and the method for glycosylglycerol is produced in cyanobacteria.And the metabolic engineering of system is carried out to photoautotrophy cyanobacteria using the method that describes of the present invention so that about 4 times to wild type of the output increased of cyanobacteria GG under salt stress.
Description
Technical field
This disclosure relates to Microbe synthesis high valuable chemicals field, specifically a kind of to be used for synthetic glycerine glucose
The engineering bacteria of glycosides and application.
Background technology
Glycosylglycerol, the material that a kind of glycerol molecule is combined with glucose molecule with glycosidic bond, according to glucose molecule
Configuration, the configuration of the position with reference to glycerol molecule and glycerol molecule be divided into it is a variety of.Wherein, nature exists at most, makees
That compress anti-molecule for natural osmotic is 2-O- (α-D-gluco-pyranosyl)-sn-glycerol (hereinafter referred to as GG), i.e.,
The main molecules that DNC wireless synthesizes under salt stress, also referred to as " compatible molecule (compatible
solutes)”.Now some researches show that GG has following purposes：（1）GG can pass through the interaction with cell intracellular macromolecular
It is a kind of stabilizer of macromolecular to maintain the Stable conformation of macromolecular, long-term available for macromoleculars such as albumen preserves, or greatly
Freeze drying protectant (Sawangwan, T., Goedl, C., Nidetzky, B., the 2010.Glucosylglycerol of molecule protein
and glucosylglycerate asenzyme stabilizers.Biotechnol.J.5,187-191.).（2）GG has
Moisture-keeping functions, can also activate epidermal cell antioxidase, such as superoxide dismutase（SOD）, glutathione peroxidase
Deng expression, can be used as a kind of cosmetic additive agent, elimination activity oxygen radical, anti-aging
（US20110207681A1）.（3）Famous white deer pure mellow wine company of Japan, due to containing GG in its wine product that ferments, so to it
Physiological and pharmacological effect has made intensive studies, and has applied for corresponding patent, specifically includes, GG can reduce blood sugar level, help
In auxiliary treatment obesity or diabetes（JP2004-331576）；Promote the generation of ossein and hyaluronic acid（JP2004-
331579）；Suppress the accumulation of neutral fats in adipocyte（JP2004-331580）；, can after face cleaning as a kind of component of face cleaning
Eliminate tight feeling（JP2004-331583）；Food, beverage additive can be used as（JP2007-262023）；Medicine can be used as to add
Agent, treats allergic dermatitis（JP2009-161564）.
The method of synthesis GG mainly includes chemical synthesis, Microbe synthesis method, Enzyme catalyzed synthesis method at present.Chemical synthesis
Aspect, since GG is there are a variety of optical isomers, 2-O- needed for acquisition (α-D-gluco-pyranosyl)-sn-glycerol productions
Rate is relatively low, and needs more purification；In terms of Microbe synthesis, although Martin Hagemann et al once reported utilization
Stenotrophomonas rhizophilastrain DSM14405 synthesize GG and secrete to extracellular, but maximum output is only about
29mg L-1(Roder et al.,2005)；In terms of Enzyme catalyzed synthesis, Japanese white deer pure mellow wine company, which once reported, utilizes malt
Sugar and glycerine are used as substrate, and using alpha-Glucosidase synthesis GG, the highest content of GG is up to 0.5% (Takenaka in its pure mellow wine
and Uchiyama,2000).2008, Bernd Nidetzky etc. were reported by the use of sucrose and glycerine as substrate, pass through sugarcane
Saccharophosphorylase（sucrose phosphorylase）Catalyze and synthesize GG.Wherein, more than 90% sucrose can be converted into GG,
Later stage is purified by adsorption chromatography etc., the purity of GG up to 98%, the purifying rate of recovery be about 70% (Goedl et al., 2008)
（US2009031872A1）.
Cyanobacteria just receives more and more attention as the photosynthetic microflora of a new generation（Angermayr, S.A. etc.
People, 2009）.In 2009, domestic and international several research groups are utilizing cyanobacteria production bio-fuel and bio-based in succession
Make a breakthrough in terms of product：Professor Fu Pengcheng of China University Of Petroleum Beijing will derive from zymomonas mobilis（Zymomonas
mobilis）Pyruvate decarboxylase and alcohol dehydrogenase gene co-expressed in DNC wireless, realize solar energy and arrive
The conversion of bio-ethanol（Yield is 5.2mmol/OD730/L/d）（And Fu, P., 2009 DexterJ.）；Primary gram of California, USA university
The research group of sharp branch school Anastasios Melis professors passes through the heterogenous expression mountain Pueraria lobota in DNC wireless
（Pueraria montana）Isoprenoid synthase gene, realize and isoprene produced in cyanobacteria（Yield is
50mg/g/d）（Lindberg, P. et al., 2009）；University of California in Los Angeles James C professors Liao have also delivered him
Newest achievement in research：Realized by genetic engineering means and isobutylaldehyde is efficiently produced in Spehococcus sp. PCC 7942（Most high yield
Measure as 6,230 μ g/L/h）（Atsumi, S. et al., 2009）, this achievement is published in Nature Biotechnology magazines
On.On March 29th, 2010, PNAS magazine rans one newest achievement in research of Ya Lisangna state universities of the U.S., that is, exist
Produced in DNC wireless and secrete free fatty（Liu, X. et al., 2010）.
Cyanobacteria（Also referred to as cyanobacteria）It is that one kind can carry out the plant type production photosynthetic prokaryotic micro-organisms of oxygen, its conduct
Industrial microorganism system synthesis GG of new generation has following advantage:（1）Cyanobacteria can absorb solar energy, fixed carbon dioxide is made
Autophyting growth is carried out for carbon source, toxigenic capacity is low, and they are rapid to strong environmental adaptability, growth；（2）Cyanobacteria from
GG can be largely synthesized under salt stress effect, is damaged for resisting salt stress caused by cell, while the synthesis and regulation and control of GG
Mechanism has also obtained extensive research（Martin Hagemann, 2011）；（3）Cyanobacteria genetic manipulation is convenient, and genetic background is clear
Clear, the gene order-checking work of numerous species has also been completed successively, this causes non-using genetic engineering means transformation cyanobacteria
It is often convenient.Wherein, cytoalgae（Synechocystis sp.）PCC6803 is the representative species of unicellular cyanobacteria, its full genome
Group sequencing was completed in 1996, was the earliest photosynthetic organism for completing genome sequencing, and the cyanobacteria of most study at present
One of, it is considered to be one of preferable pattern species（Angermayr, S.A. et al., 2009）.So changed by genetic engineering
Cyanobacteria DNC wireless is made, synthesizes the GG that there is extensive use in fields such as cosmetics, medicine, health products, food, tool
There are important research value and actual application prospect.
The content of the invention
Present invention aims at provide a kind of engineering bacteria and application for synthetic glycerine glucoside.
To achieve the above object, the technical solution adopted by the present invention is：
A kind of engineering bacteria for synthetic glycerine glucoside, engineering bacteria are GG movement systems gene and/or the negative tune of GG synthesis
Control the cyanobacteria mutant of protein gene missing.
The GG movement systems gene is in slr0529 genes, slr0530 genes, slr0531 gene in GG movement systems
One or more.
The GG movement systems gene be GG movement systems in slr0530 genes and slr0531 genes, i.e. ggtCD.It is described
Resistant maker gene is further included in cyanobacteria mutant, resistant maker gene is selected from spectinomycin resistance gene Omega fragments
(SEQ ID NO:9), kalamycin resistance gene fragment (SEQ ID NO:Or chloramphenicol resistance gene fragment (SEQ ID 10)
NO:11)。
The cyanobacteria is cytoalgae (Synechocystis sp.) PCC6803, Synechococcus (Synechococcus)
PCC7002, Synechococcus WH5701, Synechococcus CB0101, Synechococcus CB0205, Synechococcus RCC307, Synechococcus WH7803, poly- ball
Algae RS9917, Synechococcus CC9311, Synechococcus, Synechococcus WH8016, Synechococcus RS9916, Synechococcus BL107, Synechococcus
CC9902, Synechococcus WH8102, Synechococcus CC9605, Synechococcus WH8109, Synechococcus PCC7335, Acaryochloris
MBIC11017, Acaryochloris CCMEE5410, indigo plant bar algae (Cyanothece) CCY0110, blue bar algae ATCC51142,
Micro- sheath algae (Microcoleus) PCC7420, sheath silk algae (Lyngbya) PCC8106, arthrospira (Athrospira) CS-328, section
Revolve algae PCC8005 or arthrospira NIES-39.
A kind of construction method of engineering bacteria for synthetic glycerine glucoside：
1）Structure is used to knock out the plasmid that coding in cyanobacteria can absorb the movement system gene of extracellular GG；
2）Structure is used for the plasmid for knocking out the negative regulation protein gene that can suppress GG synthesis encoded in cyanobacteria；
3) by step 1）Gained plasmid, which is transformed into cyanobacteria, obtains engineering bacteria；Or by step 1）With step 2）Gained
Plasmid is successively transformed into cyanobacteria and obtains engineering bacteria.
A kind of application of engineering bacteria for synthetic glycerine glucoside, the engineering bacteria answering in synthetic glycerine glucoside
With.
Under the engineering bacteria illumination solar energy can be utilized to fix carbon dioxide growth, and under salt stress effect, can synthesize,
Accumulate and secrete glycosylglycerol.
Relational language
Cyanobacteria（Also referred to as cyanobacteria）It is a kind of photoautotrophic prokaryotic micro-organisms, it can utilize solar energy, fix two
Carbonoxide.
GG movement systems（glucosylglycerol transport system）It is that DNC wireless is used for born of the same parents
Outer GG is transported to the protein complexes of intracellular, is slr0529 genes respectively comprising three subunits（GgtB, substrate-
binding protein）, slr0530 genes（GgtC, permease protein）, slr0531 genes（GgtD, permease
protein）.Result of study shows, as long as soon as these three subunits are knocked, DNC wireless is lost extracellular GG
It is recycled to the function of intracellular（Mikkat,S.,Hagemann,M.,2000.Molecular analysis of the ggtBCD
gene cluster of Synechocystis sp.strain PCC6803encoding subunits of an ABC
transporter for osmoprotective compounds.Arch Microbiol.174,273-82.）GgpS genes
Encoding glycerol glucoside phosphate synthase (glucosylglycerol phosphate synthase), enzymatic collection born of the same parents
The first step reaction of algae PCC6803GG route of synthesis；The gene is knocked out, cell cannot synthesize GG, and sensitive to salt stress
(Marin K. et al., 1998).
One transcriptional regulation protein of ggpR gene codes, is that DNC wireless GG synthesis key gene ggpS is transcribed
Negative regulation albumen.The gene is knocked out, its Transcription inhibition for ggpS can be released so that the increase of ggpS gene transcripts content
(Klahn,S.,et al.(2010)"The gene ssl3076encodes a protein mediating the salt-
induced expression of ggpS for the biosynthesis of the compatible solute
glucosylglycerol in Synechocystis sp.strain PCC6803"J Bacteriol192(17):
p.4403-12.)。
In embodiments of the invention, carrier（vector）It is to refer to DNA fragmentation（Target gene）Be transferred to by
A kind of DNA molecular of self-replacation in person's cell.
Advantage for present invention：
The construct of synthetic glycerine glucoside of the present invention, includes the carrier of the construct, includes the construct or use
The cyanobacteria of carrier conversion, and the method for producing in cyanobacteria glycosylglycerol.And the side described using the present invention
Method carries out photoautotrophy cyanobacteria the metabolic engineering of system so that the output increased of cyanobacteria GG is to wild under salt stress
About 4 times of type.
Brief description of the drawings
Fig. 1 is the basic block diagram of plasmid pWD12 provided in an embodiment of the present invention.Wherein, respectively by cytoalgae
Mode is cascaded the fragment and Omega resistant genes of PCC6803ggtCD genes upstream and downstream about 1kb as illustrated, for striking
Except ggtCD genes.
Fig. 2 is the basic block diagram of plasmid pWD41 provided in an embodiment of the present invention.It is by ggpR genes by PCR
It is inserted into after downstream gene amplification on pMD18-T simple carriers, that resistant gene then will be blocked by digestion and be inserted into the fragment
Between.
Fig. 3 is the photo in kind of pillar Photoreactor culturing gene engineering algae strain.
Fig. 4 is the GG Yield mappings of genetic engineering algae strain under pillar Photoreactor condition of culture.A figures are that each algae strain is secreted into born of the same parents
Outer GG contents；B figures are the total GG yield of each algae strain.Wild type represent DNC wireless wild-type strain；ΔggtCD
Represent DNC wireless Δ ggtCD gene mutation strains；Δ ggtCD Δs ggpR represents cytoalgae PCC6803ggtCD and ggpR
Double-mutant strain.
Embodiment
Embodiment 1：Structure is used for the carrier for converting cyanobacteria
Sequence table information：
SEQ ID NO:1：PCC6803ggtCD gene orders.
SEQ ID NO:2：PCC6803ggpR gene orders.
SEQ ID NO:3：The sequence of primer ggtCD-up-Fwd.
SEQ ID NO:4：The sequence of primer ggtCD-up-Rev.
SEQ ID NO:5：The sequence of primer ggtCD-down-Fwd.
SEQ ID NO:6：The sequence of primer ggtCD-down-Rev.
SEQ ID NO:7：The sequence of primer ggpR-Fwd.
SEQ ID NO:8：The sequence of primer ggpR-Rev.
SEQ ID NO:9：Spectinomycin resistance gene Omega fragment sequences.
SEQ ID NO:10：Kalamycin resistance gene fragment sequence.
SEQ ID NO:11：Chloramphenicol resistance gene fragment sequence.
1st, for the structure for the plasmid pWD12 that ggtCD genes are knocked out in cyanobacteria DNC wireless genome
With ggtCD-up-Fwd（5’-GCTGCTAATGGTTATGAAGTTCCTGG-3’）And ggtCD-up-Rev（5’-
CAGTCTCTAGGGTGGGCAATATTAGATA-3’）For primer pair, PCR is carried out by template of DNC wireless genome,
And PCR product is cloned into pMD18-T carriers（Takara,Catalog No.:D101A）In, so as to obtain plasmid pWD9.With
DraI（Takara,Catalog No.:D1037A）Digested plasmid pRL57（Cai Y. et al., 1990）, recycling about 1.9kb's
Omega fragments；By plasmid pWD9 through XbaI（Takara,Catalog No.:D1093A）Digestion, with T4DNA polymerases
（Fermentas,Catalog No.:EP0061）Filling-in, recycles the carrier segments of linear pWD9；Above-mentioned two fragment is connected
To plasmid pWD11.Again with ggtCD-down-Fwd（5’-GAAGTACCATTGCCGTCATTTTGTTG-3’）And ggtCD-down-
Rev（5’-ATTACTCAGTTGGATGGTAACAGGG-3’）For primer pair, carried out by template of DNC wireless genome
PCR, PCR fragment is cloned into pMD18-T carriers, obtains plasmid pWD10；Utilize PstI（Takara,Catalog No.:
D1093A）And EcoRI（Takara,Catalog No.:D1040A）Two restriction endonucleases cut ggtCD-up from plasmid pWD11
And Omega fragments, T4DNA polymerase filling-in；With SalI（Takara,Catalog No.:D1080A）Digestion pWD10, T4DNA
Polymerase filling-in, recycles linear pWD10 carrier segments；Two fragments are connected to obtain plasmid pWD12.
Above-mentioned PCR reaction systems and condition are as follows：
50 μ L reaction systems (in system in addition to template, primer and water, other are purchased from Fermentas companies)：5μ
L10X Taq Buffer with(NH4)2SO4、4μL25mM MgCl2、2μL dNTP Mix(2.5mM each)、2.5U Taq
DNA Polymerase, 1.25U pfu DNA Polymerase, 10ng DNC wireless genomic DNA, addition two are drawn
Thing is to concentration 200nM plus water polishing to 50 μ L.
Reaction condition：95 degree, 5 minutes；(95 degree, 30 seconds of heating schedule；55 degree, 30 seconds；72 degree, 2 minutes) circulate 30 times；
72 degree, 10 minutes.
2nd, in cyanobacteria DNC wireless genome knock out ggpR genes plasmid pWD41 structure with
ggpR-Fwd（5’-TAAATCCGCCCGTTCCCTCT-3’）And ggpR-Rev（5’-GGTCTACCACAACCCGTCTG-3’）For
Primer pair, PCR (PCR reaction systems and condition are same as above) is carried out by template of DNC wireless genome, and by PCR product
It is cloned into pMD18-T simple carriers（Takara,Catalog No.:D103A）In, so as to obtain plasmid pWD37.By plasmid
PRL446 BamHI（Takara,Catalog No.:D1010A）Digestion, T4DNA polymerase filling-in, recycles the fragment of 1kb；Again
By plasmid pWD37 HpaI digestions（Takara,Catalog No.:D1064A）, recycle the fragment of 5kb；Two fragment of the above connects
Connect, so as to obtain pWD41.
Embodiment 2：The conversion of cyanobacteria and the screening of transformant
In order to knock out GG movement system gene ggtCD, DNC wireless wild-type bacteria is converted with plasmid pWD12 first
Strain, ggtCD mutant strains WD037 is obtained by antibiotic-screening；And in order to further be knocked out on the basis of ggtCD mutant strains
GgpR, then then ggtCD and ggpR double-mutant strains WD094 is obtained with plasmid pWD41 conversion ggtCD mutant strains.Detailed indigo plant is thin
Bacterium converts and screening technique is as follows：
1. plasmid pWD12 converts DNC wireless wild-type strain：
1）Take and be in exponential phase（OD730About 0.5~1.0）PCC6803 wild type frustule 10mL, centrifugation receive
Collect cell；And wash cell twice with fresh BG11 culture mediums, then cell is resuspended in 1mL BG11 culture mediums, wherein
BG11 culture mediums are 1.5g L-1NaNO3,40mg L-1K2HPO4·3H2O,36mg L-1CaCl2·2H2O,6mg L-1Citric acid,
6mg L-1Ferric citrate, 1mg L-1EDETATE DISODIUM, 20mgL-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）Above-mentioned 0.2mL cell suspensions are taken in new EP pipes, add the pWD12 expression plasmids of 2~3 μ g, are mixed, juxtaposition
In 30 DEG C, 30 μ E m-2s-1When incubation 5 is small under illumination condition.
3）By step 2）The mixture of frustule and DNA after incubation, which is coated on, is layered on BG11 tablets（Non- added with antibiotic）On
Nitrocellulose filter on, be placed in 30 DEG C, 30 μ E m-2s-1When culture 24 is small under illumination condition.Then, by nitrocellulose
Film transfer is to containing 10 μ g mL-1On the BG11 tablets of spectinomycin, and in 30 DEG C, 30 μ E m-2s-1Under conditions of continue to cultivate
About 5~7 days, picking transformant, i.e. ggtCD mutant strains WD037,10 μ g mL were added with by mutant strain in fresh-1It is grand mould
It is enriched with, is linked into after enrichment added with 10 μ g mL in the BG11 of element-1Cultivate, preserve in the liquid B G11 culture mediums of spectinomycin
It is stand-by.
2. plasmid pWD12 converts DNC wireless wild-type strain, then then with plasmid pWD41 conversion ggtCD mutation
Strain：
1）Take and be in exponential phase（OD730About 0.5~1.0）Above-mentioned ggtCD mutant strains WD037 frustule 10mL,
Cell is collected by centrifugation；And wash cell twice with fresh BG11 culture mediums, then cell is resuspended in 1mL BG11 culture mediums,
Wherein BG11 culture mediums are 1.5g L-1NaNO3,40mg L-1K2HPO4·3H2O,36mg L-1CaCl2·2H2O,6mg L-1Lemon
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）Above-mentioned 0.2mL cell suspensions are taken in new EP pipes, add the pWD41 expression plasmids of 2~3 μ g, are mixed, juxtaposition
In 30 DEG C, 30 μ E m-2s-1When incubation 5 is small under illumination condition.
3）By step 2）The mixture of frustule and DNA after incubation, which is coated on, is layered on BG11 tablets（Non- added with antibiotic）On
Nitrocellulose filter on, be placed in 30 DEG C, 30 μ E m-2s-1When culture 24 is small under illumination condition.Then, by nitrocellulose
Film transfer is to containing 10 μ g mL-1Spectinomycin and 25 μ g mL-1On the BG11 tablets of kanamycins mycin, and 30
℃、30μE m-2s-1Under conditions of continue culture about 5~7 days, picking transformant, i.e. ggtCD and ggpR double-mutant strains
WD094,10 μ g mL are added with by mutant strain in fresh-1Spectinomycin and 20 μ g mL-1It is rich in the BG11 of kanamycins mycin
Collection, is linked into after enrichment added with 10 μ g mL-1Spectinomycin and 20 μ g mL-1The liquid B G11 culture mediums of kanamycins mycin
Middle culture, preserves stand-by.
It is general that wherein mutant strain WD037 and double-mutant strain WD094 is preserved in China Committee for Culture Collection of Microorganisms
Logical microorganism center, depositary institution address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and preservation date is 2013 10
The moon 31.Mutant strain WD037 preserving numbers are CGMCC No.8428, and taxology is named as cytoalgae Synechocystis sp.；
Mutant strain WD094 preserving numbers are CGMCC No.8429, and taxology is named as cytoalgae Synechocystis sp..
Embodiment 3：GG is produced with cyanobacteria wild-type strain or the cyanobacteria mutant strain transformed through genetic engineering
1st, cyanobacteria strains culture and GG volume analysis steps：
（1）Training method one：Shaking culture.Common 500 milliliters of conical flasks, fill 300mL liquid B G11 culture mediums（Containing phase
Answer concentration antibiotic）, initial inoculation bacterial strain OD730Concentration is 0.05, in 30 DEG C, 30 μ E m-2s-1Under illumination condition, blowing air training
Support 7~8 days.
Wherein bacterial strain is respectively DNC wireless wild-type strain, mutant strain WD037, double-mutant strain WD094.
Training method two：Pillar Photoreactor culture.Simple glass pipe, pillar height 575mm, diameter 30mm, liquid amount
200mL.Initial inoculation bacterial strain OD730Concentration is 1, in 30 DEG C, 100 μ E m-2s-1Under illumination condition, lead to and contain 5%（Volume ratio）CO2's
Air is cultivated, and treats strain culturing to logarithmic growth latter stage（OD730For 7~8）, add the saturation 5M with BG11 solution allocations
NaCl solution, adjusts to the final concentration of 600mM of NaCl.
Wherein bacterial strain is respectively DNC wireless wild-type strain, mutant strain WD037, double-mutant strain WD094.
（2）After salt stress, the different strains sample 1ml of separately sampled above two mode culture when 24, then with
10000g, centrifuges 5min, respectively sucks supernatant in another EP pipes；
（3）Intracellular GG is measured：Obtained above-mentioned precipitating respectively with the resuspension of 1ml80% ethanol solutions, 65 DEG C of water-bath 4h extractings
GG, separates supernatant into 10ml centrifuge tubes with 10000g centrifugations 5min, is dried up in 55 DEG C of nitrogen, after adding pure water dissolving, dilution
Filtering carries out ion chromatography（Referring to table 1）；
（4）Extracellular GG measure：
1）Directly by（2）It is 10-20mg L that supernatant after Walk centrifugations, which is diluted to GG concentration,-1, ion color is carried out after filtering
Spectrum analysis.
2）Ion chromatography uses the ICS-3000ion-exchange chromatography system of DIONEX,
Chromatographic column isMA1analytical column.Elution requirement is：Eluted with 800mM NaOH, flow velocity 0.4ml
min-1.（Referring to table 1）
By above-mentioned gained mutant strain WD037 respectively（GgtCD mutant strains）And WD094（GgtCD and ggpR double-mutant strains）
Cultivated in pillar Photoreactor, determine the yield of GG, as a result as shown in Figure 3 and Table 1.The result shows that by gene
After engineered, the ability of cyanobacteria synthesis GG greatly improves：After knocking out GG movement system genes ggtCD, WD037GG total outputs
1.6 times, which are improved, relative to wild type (reaches 248.52mg L-1), and extracellular GG contents account for the 55% of total output；And herein
On the basis of further knock out GG synthesis negative regulation protein gene ggpR, GG total output be further increased to 433.69mg L-1, relatively
75% is further increased in WD037.
These experimental results, it was demonstrated that cyanobacteria is synthesized by photosynthesis and secretes the ability of GG；Also demonstrate and pass through
GG movement systems gene and GG synthesis negative regulation protein gene are knocked out, the yield of cyanobacteria GG can be effectively improved.
The GG yield of genetic engineering algae strain under 1. pillar condition of culture of table
Note：
Whether the salt stress time persistently rises depending on GG synthetic quantities.
It will be appreciated by persons skilled in the art that numerous change can be carried out to the present invention as shown in the specific embodiments
Become and/or modify, without departing from such as broadly described the spirit or scope of the present invention.Therefore, these embodiments are in all sides
Face is considered as illustrative and nonrestrictive.
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Claims (6)
- A kind of 1. engineering bacteria for synthetic glycerine glucoside, it is characterised in that：Engineering bacteria closes for GG movement systems gene and GG Into the cyanobacteria mutant of negative regulation protein gene missing;The GG movement systems gene be GG movement systems in slr0530 genes and slr0531 genes, i.e. ggtCD；GG synthesis negative regulation protein gene is one transcriptional regulation protein of coding, i.e. ggpR genes;The engineering bacteria is double-mutant strain WD094, and preserving number is CGMCC No.8429.
- 2. the engineering bacteria for synthetic glycerine glucoside as described in claim 1, it is characterised in that：The cyanobacteria mutant In further include resistant maker gene, resistant maker gene is selected from SEQ ID NO:Spectinomycin resistance gene Omega shown in 9 Fragment, SEQ ID NO:Kalamycin resistance gene fragment or SEQ ID NO shown in 10:Chlorampenicol resistant shown in 11 Genetic fragment.
- 3. the engineering bacteria for synthetic glycerine glucoside as described in claim 1, it is characterised in that：The cyanobacteria is collection born of the same parents Algae (Synechocystis sp.) PCC6803.
- A kind of 4. construction method of the engineering bacteria for synthetic glycerine glucoside described in claim 1, it is characterised in that：1）Structure is used to knock out the plasmid that coding in cyanobacteria can absorb the movement system gene of extracellular GG；2）Structure is used for the plasmid for the negative regulation protein gene that can suppress GG synthesis for knocking out cyanobacteria coding；3) by step 1）Gained plasmid, which is transformed into cyanobacteria, obtains engineering bacteria；Or by step 1）With step 2）Gained plasmid Successively it is transformed into cyanobacteria and obtains engineering bacteria.
- A kind of 5. application of the engineering bacteria for synthetic glycerine glucoside described in claim 1, it is characterised in that：The engineering Application of the bacterium in synthetic glycerine glucoside.
- 6. the application of the engineering bacteria for synthetic glycerine glucoside as described in claim 5, it is characterised in that：The engineering bacteria Under illumination solar energy can be utilized to fix carbon dioxide growth, and under salt stress effect, it can synthesize, accumulate and secrete glycerine glucose Glycosides.
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| CN104946618A (en) * | 2014-03-25 | 2015-09-30 | 中国科学院青岛生物能源与过程研究所 | Gel-packaged cyanobacteria cell, and packaging method and application thereof |
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| CN108977371B (en) * | 2017-05-31 | 2021-05-25 | 中国科学院青岛生物能源与过程研究所 | Cyanobacteria strain capable of being used for production of glycerol glucoside and application thereof |
| KR102232837B1 (en) * | 2017-06-12 | 2021-03-29 | 씨제이제일제당 주식회사 | Novel polypeptides having glucosylglycerol productivity and a method for producing glucosylglycerol using the same |
| CN108864218B (en) * | 2018-08-10 | 2020-09-18 | 中国科学院青岛生物能源与过程研究所 | Glycerol glucoside product, purification method and application of glycerol glucoside |
| CN108913635A (en) * | 2018-08-10 | 2018-11-30 | 中国科学院青岛生物能源与过程研究所 | A method of producing recoverin matter content during glycosylglycerol |
| CN108977402B (en) * | 2018-08-10 | 2021-01-01 | 中国科学院青岛生物能源与过程研究所 | Culture method for obtaining high-content glycerol glucoside algae cells |
| CN109627301B (en) * | 2019-02-18 | 2022-08-05 | 浙江新银象生物工程有限公司 | Development and application of Nisin solid stabilizer |
| CN112575020B (en) * | 2020-12-22 | 2022-08-26 | 天津大学 | Salt-tolerant synechococcus gene engineering bacterium capable of biologically synthesizing glycerol glucoside and construction method |
| CN114645009B (en) * | 2022-04-28 | 2023-06-30 | 中国科学院青岛生物能源与过程研究所 | Fructokinase-disabled cyanobacteria and its use for secretion and production of fructose |
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