[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

CN104651287A - Engineering bacterium for synthesizing glycosylglycerol and application thereof - Google Patents

Engineering bacterium for synthesizing glycosylglycerol and application thereof Download PDF

Info

Publication number
CN104651287A
CN104651287A CN201310598111.2A CN201310598111A CN104651287A CN 104651287 A CN104651287 A CN 104651287A CN 201310598111 A CN201310598111 A CN 201310598111A CN 104651287 A CN104651287 A CN 104651287A
Authority
CN
China
Prior art keywords
synechococcus
gene
cyanobacteria
glycosylglycerol
engineering bacteria
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201310598111.2A
Other languages
Chinese (zh)
Other versions
CN104651287B (en
Inventor
吕雪峰
杜伟
谈晓明
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Original Assignee
Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Qingdao Institute of Bioenergy and Bioprocess Technology of CAS filed Critical Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Priority to CN201310598111.2A priority Critical patent/CN104651287B/en
Publication of CN104651287A publication Critical patent/CN104651287A/en
Application granted granted Critical
Publication of CN104651287B publication Critical patent/CN104651287B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00Preparation of compounds containing saccharide radicals
    • C12P19/44Preparation of O-glycosides, e.g. glucosides
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02PCLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00Technologies relating to chemical industry
    • Y02P20/10Process efficiency
    • Y02P20/133Renewable energy sources, e.g. sunlight
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02PCLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00Technologies relating to chemical industry
    • Y02P20/50Improvements relating to the production of bulk chemicals
    • Y02P20/59Biological synthesis; Biological purification

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention relates to the field of microbiological synthesis of high valuable chemicals, specifically to an engineering bacterium for synthesizing glycosylglycerol and an application thereof. The engineering bacterium is a delection cyanobacteria mutant of GG movement system genes and/or GG synthesis negative regulatory protein genes. The engineering bacterium can fix growth of carbon dioxide by the utilization of solar energy under illumination and can synthesize, accumulate and secrete glycosylglycerol under action of salt stress. A construct for synthesizing glycosylglycerol includes a carrier of the construct, cyanobacteria which contains the construct or is converted by the use of the carrier and a method for producing glycosylglycerol from cyanobacteria. By the method, photoautophic cyanobacteria undergo systemic metabolic engineering reconstruction, thus increasing yield of cyanobacteria GG under salt stress by about four times than that of a wild type.

Description

A kind of engineering bacteria for the synthesis of glycosylglycerol and application
Technical field
The present invention relates to Microbe synthesis high valuable chemicals field, specifically a kind of engineering bacteria for the synthesis of glycosylglycerol and application.
Background technology
Glycosylglycerol, the material that a class glycerol molecule and glucose molecule are combined with glycosidic link, the configuration of the configuration according to glucose molecule, the position in conjunction with glycerol molecule and glycerol molecule is divided into multiple.Wherein, nature exists maximum, that compress anti-molecule as natural osmotic is 2-O-(α-D-gluco-pyranosyl)-sn-glycerol (hereinafter referred to as GG), namely the DNC wireless main molecules of synthesizing under salt stress, is also referred to as " compatible molecule (compatible solutes) ".Now there are some researches show; GG has following purposes: (1) GG can by maintaining macromolecular Stable conformation with macromolecular interaction in cell born of the same parents; it is a kind of macromolecular stablizer; can be used for the macromolecular long-term preservations such as albumen; or the lyophilized vaccine (Sawangwan of high molecular weight protein; T.; Goedl; C.; Nidetzky; B., 2010.Glucosylglycerol and glucosylglycerate as enzyme stabilizers.Biotechnol.J.5,187-191.).(2) GG has moisture-keeping functions, also can activate epidermic cell antioxidase, as the expression of superoxide-dismutase (SOD), Selenoperoxidase etc., can use as a kind of cosmetics additive, eliminate activity oxyradical, anti-ageing (US20110207681A1).(3) the white deer pure mellow wine company that Japan is famous, owing to containing GG in its fermented wine product, so conduct in-depth research its physiological and pharmacological effect, and applied for corresponding patent, specifically comprise, GG can reduce glucose level, contributes to assisting therapy obesity or diabetes (JP2004-331576); Promote osso-albumin and hyaluronic generation (JP2004-331579); Suppress the accumulation (JP2004-331580) of neutral fat in adipocyte; As a kind of composition of clean, tight sense (JP2004-331583) after clean, can be eliminated; Can be used as food, drink additive (JP2007-262023); Can be used as medicated premix, treatment allergic dermatitis (JP2009-161564).
The method of current synthesis GG mainly comprises chemical synthesis, Microbe synthesis method, Enzyme catalyzed synthesis method.Chemosynthesis aspect, because GG exists multiple optical isomer, obtains required 2-O-(α-D-gluco-pyranosyl)-sn-glycerol productive rate lower, and needs more purification; Microbe synthesis aspect, utilized Stenotrophomonas rhizophilastrain DSM14405 to synthesize GG although Martin Hagemann et al once reports and secrete to outside born of the same parents, production peak is only about 29mg L -1(Roder et al., 2005); Enzyme catalyzed synthesis aspect, Japanese white deer pure mellow wine company once reported and utilizes maltose and glycerine as substrate, utilized alpha-glucosidase to synthesize GG, and in its pure mellow wine, the most high-content of GG can reach 0.5% (Takenaka and Uchiyama, 2000).2008, Bernd Nidetzky etc. reported and utilizes sucrose and glycerine as substrate, catalyzes and synthesizes GG by sucrose phosphorylase (sucrose phosphorylase).Wherein, the sucrose of more than 90% can be converted into GG, and the later stage, the purity of GG can reach 98% by purifying such as adsorption chromatographies, and the purifying rate of recovery is about 70% (Goedl et al., 2008) (US2009031872A1).
Cyanobacteria is just receiving increasing concern (people such as Angermayr, S.A., 2009) as the photosynthetic microflora of a new generation.In 2009, domestic and international several research group makes a breakthrough utilizing cyanobacteria to produce in biofuel and bio-based chemical in succession: professor Fu Pengcheng of China University Of Petroleum Beijing will derive from pyruvic carboxylase and alcohol dehydrogenase gene coexpression in DNC wireless of zymomonas mobilis (Zymomonas mobilis), and (output is 5.2mmol/OD to the conversion of bio-ethanol to achieve sun power 730/ L/d) (Dexter J. and Fu, P., 2009); The research group that Univ California-Berkeley Anastasios Melis teaches is by the isoprenoid synthase gene of heterogenous expression mountain Pueraria lobota (Pueraria montana) in DNC wireless, achieve and produce isoprene (output is 50mg/g/d) (Lindberg in cyanobacteria, P. people is waited, 2009); University of California in Los Angeles James C professor Liao has also delivered their up-to-date achievement in research: (production peak is 6 to achieve High-efficient Production isobutyric aldehyde in Spehococcus sp. PCC 7942 by genetic engineering means, 230 μ g/L/h) (Atsumi, S. people is waited, 2009), this achievement is published on Nature Biotechnology magazine.On March 29th, 2010, the achievement in research that of PNAS magazine ran Ya Lisangna state university of the U.S. is up-to-date, namely produces and secretion free fatty acids people such as (, 2010) Liu, X. in DNC wireless.
Cyanobacteria (also referred to as blue-green algae) is that a class can carry out the photosynthetic prokaryotic micro-organisms of plant type product oxygen, it has following advantage as industrial microorganism system synthesis GG of new generation: (1) cyanobacteria can absorb sun power, stabilizing carbon dioxide carries out autophyting growth as carbon source, toxigenic capacity is low, and they are strong to adaptive capacity to environment, growth rapidly; (2) cyanobacteria from synthesizing GG in a large number under salt stress effect, and for resisting the damage that salt stress causes cell, the biosynthesis and regulation mechanism of GG have also been obtained and studies (Martin Hagemann, 2011) widely simultaneously; (3) cyanobacteria genetic manipulation is convenient, and genetic background is clear, and the gene order-checking work of numerous species also completes successively, and this makes to utilize genetic engineering means transformation cyanobacteria very convenient.Wherein, cytoalgae (Synechocystis sp.) PCC6803 is the representative species of unicellular cyanobacteria, its genome sequencing completed in 1996, it is the photosynthetic organism completing genome sequencing the earliest, also be one of cyanobacteria of current most study, be considered to one of desirable pattern species (people such as Angermayr, S.A., 2009).So, by genetic engineering modified cyanobacteria DNC wireless, synthesize the GG in fields such as makeup, medicine, healthcare products, food with extensive use, there is important research value and actual application prospect.
Summary of the invention
The object of the invention is to provide a kind of engineering bacteria for the synthesis of glycosylglycerol and application.
For achieving the above object, the technical solution used in the present invention is:
For the synthesis of an engineering bacteria for glycosylglycerol, engineering bacteria is the cyanobacteria mutant that GG movement system gene and/or GG synthesize negative regulation protein gene disappearance.
Described GG movement system gene is one or more in slr0529 gene in GG movement system, slr0530 gene, slr0531 gene.
Described GG movement system gene is slr0530 gene and slr0531 gene, i.e. ggtCD in GG movement system.Also comprise resistant maker gene in described cyanobacteria mutant, resistant maker gene is selected from spectinomycin resistance gene Omega fragment (SEQ ID NO:9), kalamycin resistance gene fragment (SEQ ID NO:10) or chloramphenicol resistance gene fragment (SEQ ID NO:11).
Described cyanobacteria is cytoalgae (Synechocystis sp.) PCC6803, synechococcus (Synechococcus) PCC7002, synechococcus WH5701, synechococcus CB0101, synechococcus CB0205, synechococcus RCC307, synechococcus WH7803, synechococcus RS9917, synechococcus CC9311, synechococcus, synechococcus WH8016, synechococcus RS9916, synechococcus BL107, synechococcus CC9902, synechococcus WH8102, synechococcus CC9605, synechococcus WH8109, synechococcus PCC7335, Acaryochloris MBIC11017, Acaryochloris CCMEE5410, blue bar algae (Cyanothece) CCY0110, blue bar algae ATCC51142, micro-sheath algae (Microcoleus) PCC7420, sheath silk algae (Lyngbya) PCC8106, artrospira spirulina (Athrospira) CS-328, artrospira spirulina PCC8005 or artrospira spirulina NIES-39.
A kind of construction process of the engineering bacteria for the synthesis of glycosylglycerol:
1) plasmid that can absorb the movement system gene of the outer GG of born of the same parents for knocking out coding in cyanobacteria is built;
2) plasmid for knocking out the negative regulation protein gene that GG can be suppressed to synthesize of encoding in cyanobacteria is built;
3) namely engineering bacteria is obtained by step 1) gained Plastid transformation to cyanobacteria; Or by step 1) and step 2) gained plasmid is successively transformed in cyanobacteria and namely obtains engineering bacteria.
For the synthesis of an application for the engineering bacteria of glycosylglycerol, the application of described engineering bacteria in synthetic glycerine glucoside.
Sun power stabilizing carbon dioxide can be utilized to grow under described engineering bacteria illumination, and under salt stress effect, can synthesize, accumulate and secrete glycosylglycerol.
relational language
Cyanobacteria (also referred to as blue-green algae) is the photoautotrophic prokaryotic micro-organisms of a class, and it can utilize sun power, stabilizing carbon dioxide.
GG movement system (glucosylglycerol transport system) is that DNC wireless is for being transported to the protein complexes in born of the same parents by outer for born of the same parents GG, comprise three subunits, slr0529 gene (ggtB respectively, substrate-binding protein), slr0530 gene (ggtC, permease protein), slr0531 gene (ggtD, permease protein).Result of study shows, as long as these three subunits are knocked, DNC wireless just loses the function (Mikkat be recycled to by outer for born of the same parents GG in born of the same parents, 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 glycosylglycerol phosphate synthase (glucosylglycerol phosphate synthase), the first step reaction of this enzyme catalysis DNC wireless GG route of synthesis, knock out this gene, cell can not synthesize GG, and to salt stress sensitivity (people such as Marin K., 1998).
GgpR genes encoding transcriptional regulation protein is the negative regulation albumen that DNC wireless GG synthesis key gene ggpS transcribes.Knock out this gene, its Transcription inhibition for ggpS can be removed, ggpS gene transcripts content is made to increase (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) refers to DNA molecular DNA fragmentation (goal gene) can being transferred to a kind of self-replacation in donee's cells.
The advantage that the present invention has:
The construct of synthetic glycerine glucoside of the present invention, comprises the carrier of described construct, comprises described construct or the cyanobacteria with described vector, and in cyanobacteria, produces the method for glycosylglycerol.And the method utilizing the present invention to describe carries out the metabolic engineering of system to photoautotrophy cyanobacteria, make the output increased of cyanobacteria GG under salt stress to about 4 times of wild-type.
Accompanying drawing explanation
The basic block diagram of the plasmid pWD12 that Fig. 1 provides for the embodiment of the present invention.Wherein, the fragment and the Omega resistant gene that respectively DNC wireless ggtCD gene upstream and downstream are about 1kb are cascaded according to diagramatic way, for knocking out ggtCD gene.
The basic block diagram of the plasmid pWD41 that Fig. 2 provides for the embodiment of the present invention.It inserts on pMD18-T simple carrier by PCR by after the gene amplification of ggpR gene upstream and downstream, then cut by enzyme and inserted in the middle of this fragment by that resistant gene of card.
Fig. 3 is the photo in kind of pillar photoreactor culturing gene engineering algae strain.
Fig. 4 is the GG Yield mapping of genetically engineered algae strain under pillar photoreactor culture condition.A figure is the GG content that each algae strain is secreted into outside born of the same parents; B figure is the total GG output of each algae strain.Wild type represents DNC wireless wild type strain; Δ ggtCD represents DNC wireless Δ ggtCD transgenation strain; Δ ggtCD Δ ggpR represents cytoalgae PCC6803ggtCD and ggpR double-mutant strain.
Embodiment
Embodiment 1: build the carrier for transforming cyanobacteria
Sequence table information:
SEQ ID NO:1:PCC6803ggtCD gene order.
SEQ ID NO:2:PCC6803ggpR gene order.
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 sequence.
SEQ ID NO:10: kalamycin resistance gene fragment sequence.
SEQ ID NO:11: chloramphenicol resistance gene fragment sequence.
1, for knocking out the structure of the plasmid pWD12 of ggtCD gene in cyanobacteria DNC wireless genome
With ggtCD-up-Fwd(5 '-GCTGCTAATGGTTATGAAGTTCCTGG-3 ') and ggtCD-up-Rev(5 '-CAGTCTCTAGGGTGGGCAATATTAGATA-3 ') be primer pair, with DNC wireless genome for template carries out PCR, and PCR primer is cloned into pMD18-T carrier (Takara, Catalog No.:D101A) in, thus obtain plasmid pWD9.With DraI(Takara, Catalog No.:D1037A) people such as digested plasmid pRL57(Cai Y., 1990), reclaim the Omega fragment of about 1.9kb; By plasmid pWD9 through XbaI(Takara, Catalog No.:D1093A) enzyme cuts, fills, reclaim the carrier segments of linear pWD9 with T4DNA polysaccharase (Fermentas, Catalog No.:EP0061); Above-mentioned two fragments are connected and obtain plasmid pWD11.Again with ggtCD-down-Fwd(5 '-GAAGTACCATTGCCGTCATTTTGTTG-3 ') and ggtCD-down-Rev(5 '-ATTACTCAGTTGGATGGTAACAGGG-3 ') be primer pair, with DNC wireless genome for template carries out PCR, PCR fragment is cloned in pMD18-T carrier, obtains plasmid pWD10; Utilize PstI(Takara, Catalog No.:D1093A) and EcoRI(Takara, Catalog No.:D1040A) two restriction endonucleases cut ggtCD-up and Omega fragment from plasmid pWD11, T4DNA polysaccharase fills; With SalI(Takara, Catalog No.:D1080A) enzyme cuts pWD10, and T4DNA polysaccharase fills, and reclaims linear pWD10 carrier segments; Two fragments are connected and obtains plasmid pWD12.
Above-mentioned PCR reaction system and condition as follows:
50 μ L reaction systems (in system except template, primer and water, other are all purchased from Fermentas company): 5 μ L 10X Taq Buffer with (NH4) 2sO 4, 4 μ L 25mM MgCl 2, 2 μ L dNTP Mix (2.5mM each), 2.5U Taq DNA Polymerase, 1.25U pfu DNA Polymerase, 10ng DNC wireless genomic dna, add two primers to concentration 200nM, add water polishing to 50 μ L.Reaction conditions: 95 degree, 5 minutes; Heating schedule (95 degree, 30 seconds; 55 degree, 30 seconds; 72 degree, 2 minutes) circulate 30 times; 72 degree, 10 minutes.
2, for knocking out the structure of the plasmid pWD41 of ggpR gene with ggpR-Fwd(5 '-TAAATCCGCCCGTTCCCTCT-3 ' in cyanobacteria DNC wireless genome) and ggpR-Rev(5 '-GGTCTACCACAACCCGTCTG-3 ') be primer pair, with DNC wireless genome for template carries out PCR (PCR reaction system and condition the same), and PCR primer is cloned into pMD18-T simple carrier (Takara, Catalog No.:D103A) in, thus obtain plasmid pWD37.By plasmid pRL446 BamHI(Takara, Catalog No.:D1010A) enzyme cuts, and T4DNA polysaccharase fills, and reclaims the fragment of 1kb; Again plasmid pWD37 HpaI enzyme is cut (Takara, Catalog No.:D1064A), reclaim the fragment of 5kb; Above two fragments connect, thus obtain pWD41.
Embodiment 2: the conversion of cyanobacteria and the screening of transformant
In order to knock out GG movement system gene ggtCD, first transforming DNC wireless wild type strain with plasmid pWD12, obtaining ggtCD mutant strain WD037 by antibiotic-screening; And in order to knock out ggpR on the basis of ggtCD mutant strain further, then transform ggtCD mutant strain with plasmid pWD41 subsequently, obtain ggtCD and ggpR double-mutant strain WD094.Detailed cyanobacteria transform and screening method as follows:
1. plasmid pWD12 transforms DNC wireless wild type strain:
1) get and be in logarithmic phase (OD 730be about 0.5 ~ 1.0) PCC6803 wild-type frustule 10mL, centrifugal collecting cell; And with fresh BG11 substratum washed cell twice, then cell is resuspended in 1mL BG11 substratum, wherein BG11 substratum is 1.5g L -1naNO 3, 40mg L -1k 2hPO 43H 2o, 36mg L -1caCl 22H 2o, 6mg L -1citric acid, 6mg L -1ferric ammonium citrate, 1mg L -1eDETATE DISODIUM, 20mgL -1naCO 3, 2.9mg L -1h 3bO 3, 1.8mg L -1mnCl 24H 2o, 0.22mg L -1znSO 47H 2o, 0.39mg L -1naMoO 42H 2o, 0.079mg L -1cuSO 45H 2o and 0.01mg L -1coCl 26H 2o.
2) get above-mentioned 0.2mL cell suspension in new EP pipe, add the pWD12 expression plasmid of 2 ~ 3 μ g, mixing, and be placed in 30 DEG C, 30 μ E m -2s -1incubation 5 hours under illumination condition.
3) by step 2) mixture of frustule after incubation and DNA coats on the nitrocellulose filter that is layered on BG11 flat board (non-added with antibiotic), and is placed in 30 DEG C, 30 μ E m -2s -1cultivate 24 hours under illumination condition.Then, nitrocellulose filter is transferred to containing 10 μ g mL -1on the BG11 flat board of spectinomycin, and at 30 DEG C, 30 μ E m -2s -1condition under continue cultivate about 5 ~ 7 days, picking transformant, i.e. ggtCD mutant strain WD037, be added with 10 μ g mL by mutant strain at fresh -1enrichment in the BG11 of spectinomycin, is linked into after enrichment and is added with 10 μ g mL -1cultivate in the liquid B G11 substratum of spectinomycin, preserve stand-by.
2. plasmid pWD12 transforms DNC wireless wild type strain, then transforms ggtCD mutant strain with plasmid pWD41 subsequently:
1) get and be in logarithmic phase (OD 730be about 0.5 ~ 1.0) above-mentioned ggtCD mutant strain WD037 frustule 10mL, centrifugal collecting cell; And with fresh BG11 substratum washed cell twice, then cell is resuspended in 1mL BG11 substratum, wherein BG11 substratum is 1.5g L -1naNO 3, 40mg L -1k 2hPO 43H 2o, 36mg L -1caCl 22H 2o, 6mg L -1citric acid, 6mg L -1ferric ammonium citrate, 1mg L -1eDETATE DISODIUM, 20mg L -1naCO 3, 2.9mg L -1h 3bO 3, 1.8mg L -1mnCl 24H 2o, 0.22mg L -1znSO 47H 2o, 0.39mg L -1naMoO 42H 2o, 0.079mg L -1cuSO 45H 2o and 0.01mg L -1coCl 26H 2o.
2) get above-mentioned 0.2mL cell suspension in new EP pipe, add the pWD41 expression plasmid of 2 ~ 3 μ g, mixing, and be placed in 30 DEG C, 30 μ E m -2s -1incubation 5 hours under illumination condition.
3) by step 2) mixture of frustule after incubation and DNA coats on the nitrocellulose filter that is layered on BG11 flat board (non-added with antibiotic), and is placed in 30 DEG C, 30 μ E m -2s -1cultivate 24 hours under illumination condition.Then, nitrocellulose filter is transferred to containing containing 10 μ g mL -1spectinomycin and 25 μ g mL -1on the BG11 flat board of kantlex mycin, and at 30 DEG C, 30 μ E m -2s -1condition under continue cultivate about 5 ~ 7 days, picking transformant, i.e. ggtCD and ggpR double-mutant strain WD094, be added with 10 μ g mL by mutant strain at fresh -1spectinomycin and 20 μ g mL -1enrichment in the BG11 of kantlex mycin, is linked into after enrichment and is added with 10 μ g mL -1spectinomycin and 20 μ g mL -1cultivate in the liquid B G11 substratum of kantlex mycin, preserve stand-by.
Wherein mutant strain WD037 and double-mutant strain WD094 is all preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and depositary institution address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and preservation date is on October 31st, 2013.Mutant strain WD037 preserving number is CGMCC No.8428, taxonomy called after cytoalgae Synechocystis sp.; Mutant strain WD094 preserving number is CGMCC No.8429, taxonomy called after cytoalgae Synechocystis sp..
Embodiment 3: produce GG with cyanobacteria wild type strain or through genetic engineering modified cyanobacteria mutant strain
1, cyanobacteria strains is cultivated and GG volume analysis step:
(1) training method one: shake-flask culture.Common 500 milliliters of Erlenmeyer flasks, dress 300mL liquid B G11 substratum (containing respective concentration microbiotic), initial inoculation bacterial strain OD 730concentration is 0.05, at 30 DEG C, 30 μ E m -2s -1under illumination condition, blowing air is cultivated 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 is cultivated.Simple glass pipe, post height 575mm, diameter 30mm, liquid amount 200mL.Initial inoculation bacterial strain OD 730concentration is 1, at 30 DEG C, 100 μ E m -2s -1under illumination condition, logical containing 5%(volume ratio) CO 2air cultivate, treat that strain culturing is to logarithmic growth (OD in latter stage 730be 7 ~ 8), add the saturated 5M NaCl solution by BG11 solution allocation, being adjusted to NaCl final concentration is 600mM.
Wherein bacterial strain is respectively DNC wireless wild type strain, mutant strain WD037, double-mutant strain WD094.
(2) after salt stress, sample the different strains sample 1ml that above-mentioned two kinds of modes are cultivated every 24 time respectively, then with 10000g, centrifugal 5min, sucks supernatant in another EP pipe respectively;
(3) in born of the same parents, GG measures: use 1ml80% ethanolic soln resuspended respectively above-mentioned being precipitated, 65 DEG C of water-bath 4h extracting GG, are separated supernatant in 10ml centrifuge tube with the centrifugal 5min of 10000g, dry up in 55 DEG C of nitrogen, after adding pure water dissolving, dilute filtration carries out ion chromatography (see table 1);
(4) the outer GG of born of the same parents measures:
1) directly by the (2) Walk centrifugal after supernatant to be diluted to GG concentration be 10-20mg L -1, after filtration, carry out ion chromatography.
2) ion chromatography adopts the ICS-3000ion-exchange chromatography system of DIONEX, and chromatographic column is mA1analytical column.Elution requirement is: with 800mM NaOH wash-out, flow velocity is 0.4ml min -1.(see table 1)
By the above-mentioned WD037(ggtCD of gained mutant strain respectively mutant strain) and WD094(ggtCD and ggpR double-mutant strain) cultivate in pillar photoreactor, determine the output of GG, result is as shown in Figure 3 and Table 1.Result shows, after genetic engineering modified, the ability of cyanobacteria synthesis GG significantly improves: after knocking out GG movement system gene ggtCD, WD037GG ultimate production improves 1.6 times relative to wild-type and (reaches 248.52mg L -1), and the outer GG content of born of the same parents accounts for 55% of ultimate production; And knocking out GG synthesis negative regulation protein gene ggpR on this basis further, GG ultimate production brings up to 433.69mg L further -1, further increase 75% relative to WD037.
These experimental results, are demonstrated cyanobacteria and are synthesized by photosynthesis and the ability of secreting GG; Also demonstrating by knocking out GG movement system gene and GG synthesis negative regulation protein gene, effectively can improve the output of cyanobacteria GG.
The GG output of genetically engineered algae strain under table 1. pillar culture condition
Note:
The salt stress time depends on whether GG resultant quantity continues to rise.
Those skilled in the art will appreciate that and can carry out numerous change and/or modification to the present invention as shown in the specific embodiments, and do not deviate from as broadly described the spirit or scope of the present invention.Therefore, these embodiments be regarded as illustrative in all respects and nonrestrictive.Reference
Sawangwan,T.,Goedl,C.,Nidetzky,B.,2010.Glucosylglycerol and glucosylglycerate as enzyme stabilizers.Biotechnol.J.5,187-191.
Angermayr,S.A.,et al.(2009).“Energy biotechnology with cyanobacteria”, Curr Opin Biotechnol20(3):257-263.
Atsumi,S.,et al.(2009).“Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde”, Nat.Biotechnol.27:1177-1180.
Cai Y.and Wolk C.(1990)“Use of a conditionally lethal gene in Anabaena sp.strain PCC7120to select for double recombinants and to entrap insertion sequences.” J.Bacteriol172:3138
Cheng J.B.and Russell D.W.(2004)“Mammalian Wax Biosynthesis:I.IDENTIFICATION OF TWO FATTY ACYL-COENZYME A REDUCTASES WITH DIFFERENT SUBSTRATE SPECIFICITIES AND TISSUE DISTRIBUTIONS.” J.Biol.Chem.279:37789-37797.
Dexter,J.and Fu,P.(2009).“Metabolic engineering of cyanobacteria for ethanol production”, Energy&Environmental Science2(8):857-864.
Keasling,J.D.,et al.(2007).“Production of fatty acids and derivatives thereof”,International Patent WO/2007/136,762.
Lee,S.K.,et al.(2008).“Metabolic engineering of microorganisms for biofuels production:from bugs to synthetic biology to fuels”, CurrOpin Biotechnol19(6):556-563.
Lindberg,P.,et al.(2009).“Engineering a platform for photosynthetic isoprene production in cyanobacteria,using Synechocystis as the model organism”, Metab Eng.
Liu,X.,et al.(2010)."Production and secretion of fatty acids in genetically engineered cyanobacteria." Proc Natl Acad Sci USA.
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.
Marin,K.,et al.(1998)."The ggpS gene from Synechocystis sp.strain PCC6803encoding glucosyl-glycerol-phosphate synthase is involved in osmolyte synthesis", J Bacteriol180(18):p.4843-9.
Pengcheng,F.(2009).“Genome-scale modeling of Synechocystis sp.PCC6803and prediction of pathway insertion”, Journal of Chemical Technology&Biotechnology84(4):473-483.
Steen,E.J.,et al.(2010).“Microbial production of fatty-acid-derived fuels and chemicals from plant biomass”, Nature463(7280):559-562.
Williams J.G.K.(1988)“Construction of specific mutations in photosystem II photosynthetic reaction center by genetic engineering methods in Synechocystis6803.” Methods in Enzymology167:766-778
The people such as Gao Hong (2007) " structure of DNC wireless cupric ion abduction delivering platform " hydrobiont journal240-244.
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。

Claims (8)

1. for the synthesis of an engineering bacteria for glycosylglycerol, it is characterized in that: engineering bacteria is the cyanobacteria mutant that GG movement system gene and/or GG synthesize negative regulation protein gene disappearance.
2., by the engineering bacteria for the synthesis of glycosylglycerol according to claim 1, it is characterized in that: described GG movement system gene is one or more in slr0529 gene in GG movement system, slr0530 gene, slr0531 gene.
3., by the engineering bacteria for the synthesis of glycosylglycerol according to claim 1, it is characterized in that: described GG movement system gene is slr0530 gene and slr0531 gene, i.e. ggtCD in GG movement system.
4. by the engineering bacteria for the synthesis of glycosylglycerol according to claim 1, it is characterized in that: also comprise resistant maker gene in described cyanobacteria mutant, resistant maker gene is selected from spectinomycin resistance gene Omega fragment (SEQ ID NO:9), kalamycin resistance gene fragment (SEQ ID NO:10) or chloramphenicol resistance gene fragment (SEQ ID NO:11).
5. by the engineering bacteria for the synthesis of glycosylglycerol according to claim 1, it is characterized in that: described cyanobacteria is cytoalgae (Synechocystis sp.) PCC6803, synechococcus (Synechococcus) PCC7002, synechococcus WH5701, synechococcus CB0101, synechococcus CB0205, synechococcus RCC307, synechococcus WH7803, synechococcus RS9917, synechococcus CC9311, synechococcus, synechococcus WH8016, synechococcus RS9916, synechococcus BL107, synechococcus CC9902, synechococcus WH8102, synechococcus CC9605, synechococcus WH8109, synechococcus PCC7335, Acaryochloris MBIC11017, Acaryochloris CCMEE5410, blue bar algae (Cyanothece) CCY0110, blue bar algae ATCC51142, micro-sheath algae (Microcoleus) PCC7420, sheath silk algae (Lyngbya) PCC8106, artrospira spirulina (Athrospira) CS-328, artrospira spirulina PCC8005 or artrospira spirulina NIES-39.
6. a construction process for the engineering bacteria for the synthesis of glycosylglycerol according to claim 1, is characterized in that:
1) plasmid that can absorb the movement system gene of the outer GG of born of the same parents for knocking out coding in cyanobacteria is built;
2) plasmid of the negative regulation protein gene that GG can be suppressed to synthesize for knocking out cyanobacteria coding is built;
3) namely engineering bacteria is obtained by step 1) gained Plastid transformation to cyanobacteria; Or by step 1) and step 2) gained plasmid is successively transformed in cyanobacteria and namely obtains engineering bacteria.
7. an application for the engineering bacteria for the synthesis of glycosylglycerol according to claim 1, is characterized in that: the application of described engineering bacteria in synthetic glycerine glucoside.
8., by the application of the engineering bacteria for the synthesis of glycosylglycerol according to claim 7, it is characterized in that:
Sun power stabilizing carbon dioxide can be utilized to grow under described engineering bacteria illumination, and under salt stress effect, can synthesize, accumulate and secrete glycosylglycerol.
CN201310598111.2A 2013-11-22 2013-11-22 A kind of engineering bacteria and application for synthetic glycerine glucoside Active CN104651287B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201310598111.2A CN104651287B (en) 2013-11-22 2013-11-22 A kind of engineering bacteria and application for synthetic glycerine glucoside

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201310598111.2A CN104651287B (en) 2013-11-22 2013-11-22 A kind of engineering bacteria and application for synthetic glycerine glucoside

Publications (2)

Publication Number Publication Date
CN104651287A true CN104651287A (en) 2015-05-27
CN104651287B CN104651287B (en) 2018-04-27

Family

ID=53242938

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201310598111.2A Active CN104651287B (en) 2013-11-22 2013-11-22 A kind of engineering bacteria and application for synthetic glycerine glucoside

Country Status (1)

Country Link
CN (1) CN104651287B (en)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN105342874A (en) * 2015-11-25 2016-02-24 珠海市时代经典化妆品有限公司 Composition with skin tightening and anti-aging functions and preparation of composition with skin tightening and anti-aging functions
CN108864218A (en) * 2018-08-10 2018-11-23 中国科学院青岛生物能源与过程研究所 Purification process and the application of a kind of glycerol-glucose glycoside product and glycosylglycerol
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
CN108977371A (en) * 2017-05-31 2018-12-11 中国科学院青岛生物能源与过程研究所 It can be used for cyanobacteria strains and its application of glycosylglycerol production
CN108977402A (en) * 2018-08-10 2018-12-11 中国科学院青岛生物能源与过程研究所 A kind of cultural method obtaining high-content glycosylglycerol frustule
WO2018230953A1 (en) * 2017-06-12 2018-12-20 씨제이제일제당 (주) Novel polypeptide having activity of producing glucosylglycerol and glucosylglycerol production method using same
CN109627301A (en) * 2019-02-18 2019-04-16 浙江新银象生物工程有限公司 Nisin Pickering agent exploitation and application
CN112575020A (en) * 2020-12-22 2021-03-30 天津大学 Salt-tolerant synechococcus gene engineering bacterium capable of biologically synthesizing glycerol glucoside and construction method
CN114645009A (en) * 2022-04-28 2022-06-21 中国科学院青岛生物能源与过程研究所 Fructokinase-disabled cyanobacteria and application thereof in secretion and fructose production

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
STEFAN MIKKAT等: "Molecular analysis of the ggtBCD gene cluster of Synechocystis sp. strain PCC6803 encoding subunits of an ABC transporter for osmoprotective compounds", 《ARCH MICROBIOL》 *
STEPHAN KLäHN等: "The Gene ssl3076 Encodes a Protein Mediating the Salt-Induced Expression of ggpS for the Biosynthesis of the Compatible Solute Glucosylglycerol in Synechocystis sp. Strain PCC 6803▽§", 《JOURNAL OF BACTERIOLOGY》 *
XIAOMING TAN等: "Photosynthetic and extracellular production of glucosylglycerol by genetically engineered and gel-encapsulated cyanobacteria", 《APPL MICROBIOL BIOTECHNOL》 *
葛平平等: "产乙醇基因工程集胞藻的盐胁迫响应", 《过程工程学报》 *
马培珍等: "ggpS 基因过表达对集胞藻PCC 6803 甘油葡萄糖苷和甘油合成的影响", 《生物工程学报》 *
齐凤霞等: "集胞藻PCC6803 高效基因表达平台的构建与评价", 《生物工程学报》 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN105342874A (en) * 2015-11-25 2016-02-24 珠海市时代经典化妆品有限公司 Composition with skin tightening and anti-aging functions and preparation of composition with skin tightening and anti-aging functions
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
CN108977371A (en) * 2017-05-31 2018-12-11 中国科学院青岛生物能源与过程研究所 It can be used for cyanobacteria strains and its application of glycosylglycerol production
WO2018230953A1 (en) * 2017-06-12 2018-12-20 씨제이제일제당 (주) Novel polypeptide having activity of producing glucosylglycerol and glucosylglycerol production method using same
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
CN108977402A (en) * 2018-08-10 2018-12-11 中国科学院青岛生物能源与过程研究所 A kind of cultural method obtaining high-content glycosylglycerol frustule
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
CN108864218A (en) * 2018-08-10 2018-11-23 中国科学院青岛生物能源与过程研究所 Purification process and the application of a kind of glycerol-glucose glycoside product and glycosylglycerol
CN109627301A (en) * 2019-02-18 2019-04-16 浙江新银象生物工程有限公司 Nisin Pickering agent exploitation and application
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
CN112575020A (en) * 2020-12-22 2021-03-30 天津大学 Salt-tolerant synechococcus gene engineering bacterium capable of biologically synthesizing glycerol glucoside and construction method
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
CN114645009A (en) * 2022-04-28 2022-06-21 中国科学院青岛生物能源与过程研究所 Fructokinase-disabled cyanobacteria and application thereof in secretion and fructose production
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

Also Published As

Publication number Publication date
CN104651287B (en) 2018-04-27

Similar Documents

Publication Publication Date Title
CN104651287B (en) A kind of engineering bacteria and application for synthetic glycerine glucoside
EP2598630B1 (en) Novel bacteria and methods of use thereof
CN105916976A (en) Carbohydrate-enriched recombinant microorganisms
CN101173308A (en) Method for ferment for producing adenomethionine with genetic engineering bacterium
CN106520715B (en) A kind of short-chain dehydrogenase and its gene, recombinant expression carrier, genetic engineering bacterium and its application in the synthesis of astaxanthin chiral intermediate
CN104093836A (en) Hydrocarbon synthase gene, and use thereor
CN104046586B (en) One strain gene engineering bacterium and the application in producing (2R, 3R)-2,3-butanediol thereof
CN114525214B (en) Construction method and application of engineering probiotics
CN101748069A (en) recombinant blue-green algae
CN107257851A (en) Positive influences are natural or combination of bacterial chaperonin of physiology of eukaryotic of engineering
CN105801675B (en) A kind of High-activity chitosanase control gene csn and the method using gene production High-activity chitosanase
CN105255951B (en) A method of Alcohol Production efficiency is improved by overexpression HAC1 genes
CN104204206B (en) A kind of method for producing butanol
CN102517303A (en) Recombination blue-green alga for producing lactic acid as well as preparation method and applications thereof
CN106754979A (en) A kind of gene of long-chain fat acid transporter of regulation and control candida tropicalis and its application
CN102952818B (en) For improving construct and the method for fatty alcohol yield in cyanobacteria
CN114736918B (en) Recombinant escherichia coli for producing salidroside by integrated expression and application thereof
KR101758910B1 (en) Recombinant Microorganisms Producing Butanol and Method for Preparing Butanol Using the Same
CN116731886A (en) Engineering bacterium for producing glycosylated astaxanthin as well as construction method and application thereof
Laptev et al. New recombinant strains of the yeast Yarrowia lipolytica with overexpression of the aconitate hydratase gene for the obtainment of isocitric acid from rapeseed oil
CN104403956B (en) The structure of xylitol high temperature high-yielding engineering bacterial strain and application
CN109929853B (en) Application of thermophilic bacteria source heat shock protein gene
Richard et al. C1-Proteins Prospect for Production of Industrial Proteins and Protein-Based Materials from Methane
CN104830851A (en) Recombinant bacterium of formate dehydrogenase and application of recombinant bacterium
CN102311966B (en) For the synthesis of the construct of fatty alcohol, carrier, cyanobacteria, and the method for producing fatty alcohol in cyanobacteria

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant