CN109370972A - A kind of acetobacter engineering bacteria and its application - Google Patents
A kind of acetobacter engineering bacteria and its application Download PDFInfo
- Publication number
- CN109370972A CN109370972A CN201811375476.8A CN201811375476A CN109370972A CN 109370972 A CN109370972 A CN 109370972A CN 201811375476 A CN201811375476 A CN 201811375476A CN 109370972 A CN109370972 A CN 109370972A
- Authority
- CN
- China
- Prior art keywords
- acetobacter
- gene
- engineering bacteria
- plasmid
- homologous recombination
- 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.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/42—Hydroxy-carboxylic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/44—Polycarboxylic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/62—Carboxylic acid esters
- C12P7/625—Polyesters of hydroxy carboxylic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/99—Oxidoreductases acting on the CH-OH group of donors (1.1) with other acceptors (1.1.99)
- C12Y101/9901—Glucose dehydrogenase (acceptor) (1.1.99.10)
Abstract
The present invention relates to a kind of acetobacter engineering bacteria and its applications, belong to gene engineering technology field.The present invention successively contains acetobacter gdh homologous recombination forearm, marker gene and gdh homologous recombination postbrachium, carrier by building gene knockout plasmid, the knockout plasmid；It successful gene knockout plasmid will be constructed is transferred to acetobacter electricity and turn competent cell, suicide plasmid is using homologous recombination twice by gene knockout, first time homologous recombination plasmid is from entering and be integrated on chromosome, second of homologous recombination plasmid is detached from from chromosome with the gene for knocking out primer amplification, i.e., gene knockout successfully obtains clpp gene degerming.Film combination glucose dehydrogenase gene that the present invention obtains knock out bacterium can in glycerol and glucose mixed culture medium well-grown, acetobacter toxigenic capacity is greatly saved, is of great significance in biocatalyst application field.
Description
Technical field
The present invention relates to a kind of acetobacter engineering bacteria and its applications, belong to gene engineering technology field.
Background technique
3- hydracrylic acid is a kind of emerging high added value platform chemicals, can synthesize a variety of important chemical substances, such as
The poly- 3- hydroxy propionate of malonic acid, acrylic acid and high intensity, high thermal stability and biodegradable properties, by american energy
Portion is classified as 12 kinds of most potential one of chemical products on our times.
Acetobacter Acetobacter sp.CGMCC 8142 is that the screening of this team obtains, and has efficient catalysis 1,3-
Propylene glycol synthesizes the ability of 3-HP, is a kind of efficient biocatalyst, however finds in the course of the research, acetobacter
The growth performance in the culture medium (glycerol and glucose) for obtaining efficient catalytic performance of CGMCC 8142 is poor, and glucose is easily by vinegar
Acidfast bacilli film combination glucose dehydrogenase (GDH) is oxidized to gluconic acid, reduces rapidly culture solution pH, limits the benefit of glycerol
With, it is unfavorable to thalli growth and catalysis, limit the possibility of its further expansion production.And the biology of current Dichlorodiphenyl Acetate bacillus
Amount culture focuses primarily upon on medium optimization, and raising acetobacter, which is transformed, by genetic engineering there is no report to the utilization rate of carbon source
Road.
Summary of the invention
It is in acetobacter Acetobacter the first purpose of the invention is to provide a kind of acetobacter engineering bacteria
Glucose dehydrogenase gene has been knocked out on the genome of sp.CGMCC8142.
The Acetobacter sp.CGMCC 8142 is quoted from Li J, Zong H, Zhuge B, et
al.Immobilization of Acetobacter sp.CGMCC 8142for efficient biocatalysis of
1,3-propanediol to 3-hydroxypropionic acid[J].Biotechnology&Bioprocess
Engineering, 2016,21 (4): 523-530 was preserved in China typical culture collection center on September 9th, 2013,
Deposit number is CCTCC NO:8142, and preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
In one embodiment of the invention, the nucleotide sequence of the glucose dehydrogenase gene such as SEQ ID
Shown in NO.1.
A second object of the present invention is to provide the construction methods of above-mentioned acetobacter engineering bacteria, which comprises
(1) gene knockout plasmid is constructed；
(2) gene knockout plasmid is converted to Acetobacter sp. competent cell.
In one embodiment of the invention, the gene knockout plasmid is using pK18mobSacB as carrier, and expression contains
Before the film combination glucose dehydrogenase gene in 8142 source Acetobacter sp.CGMCC shown in SEQ ID NO.2 is homologous
The film combination glucose dehydrogenase in 8142 source Acetobacter sp.CGMCC shown in arm gene and the NO.3 of ID containing SEQ
DNA homolog postbrachium gene.
Third object of the present invention is to provide application of the above-mentioned acetobacter engineering bacteria in catalysis biochemical reaction.
Fourth object of the present invention be to provide above-mentioned acetobacter engineering bacteria production 3- hydracrylic acid, acrylic acid,
Application in 1,3-PD, malonic acid or poly- 3- hydracrylic acid.
Fifth object of the present invention is to provide above-mentioned acetobacter engineering bacteria food or field of fodder application.
Sixth object of the present invention is to provide above-mentioned acetobacter engineering bacterias in the additive for preparing food or feed
Or the application in terms of preservative.
7th purpose of the invention is to provide above-mentioned acetobacter engineering bacteria in the application of chemical industry or pharmaceutical field.
8th purpose of the invention be to provide above-mentioned acetobacter engineering bacteria preparation biodegradable thermoplastic's polyester,
Application in terms of insecticide, coating, adhesive, plasticizer or emulsifier.
It is that the present invention obtains the utility model has the advantages that the present invention constructs a kind of acetobacter engineering bacteria Acetobacter sp. △
The bacterial strain is placed in most suitable catalytic performance culture medium when cultivating by gdh, eliminates saccharic acid caused by glucose dehydrogenase is catalyzed
Accumulation, to promote utilization of the thallus to glycerol.After strain culturing 72h, OD600Value improves 1.72 times compared with wild mushroom,
PH also no longer rapid decrease, more adaptation large-scale application.
Detailed description of the invention
Fig. 1: knockout carrier constructs schematic diagram.
Fig. 2: gene knockout schematic diagram, the black segment in figure are quasi- to knock out gene gdh segment.
Electrophoretogram before and after Fig. 3: gdh gene knockout, swimming lane 1 are molecular weight standard, and swimming lane 2 is the gdh gene before knocking out, swimming
Road 3 is the gdh gene after knocking out.
The growth change figure of 8142 engineering bacteria of Acetobacter sp.CGMCC after Fig. 4: gdh gene knockout.
The growth change figure of Acetobacter indonesiensis engineering bacteria after Fig. 5: gdh gene knockout.
Specific embodiment
(1) culture medium
Seed culture medium: glycerol 12g/L, peptone 10g/L, KH2PO41g/L, MgSO4·7H2O 1g/L。
Most suitable catalytic performance culture medium: glycerol 12g/L, glucose 8g/L, peptone 10g/L, KH2PO41g/L,
MgSO4·7H2O 1g/L；Agar powder 2% is added when preparing solid medium.
Kanamycins working concentration: 50 μ g/mL.
It in following embodiments, explains more, is all using conventional experimental methods of molecular biology.
The building of 1 gene knockout plasmid of embodiment
(1) the 16S rDNA sequence homology with Acetobacter sp.CGMCC 8142 is searched in ncbi database
The whole genome sequence of higher Acetobacter indonesiensis 5H-1, according to Acetobacter
The film combination glucose dehydrogenase gdh gene order (as shown in SEQ ID NO.1) of indonesiensis 5H-1 designs gene
Homology arm primer and gene knockout verifying primer and resistance marker primer are knocked out, as shown in table 1.Wherein homology arm amplimer expands
Increasing production object is about 1500bp, and resistance marker amplified production is about 900bp, and knocking out verifying amplified production is about 2000bp.
Primer is verified after 1 gdh gene knockout homology arm amplimer of table, resistance marker primer and knockout
Extraction 8142 genomic DNA of Acetobacter sp.CGMCC is template, respectively with same containing homologous recombination connection
Primer gdh1-F1, gdh1-R1 and gdh2-F1 of source arm and restriction enzyme site, gdh2-R1 amplification gene knock out homology arm.
PCR system: 0.5 5 μ L of μ L, 10 × buffer of Extaq enzyme, template 1 μ L, dNTP 4 μ L, each 1.5 μ L of primer,
ddH2O 36.5μL。
RCR program: 94 DEG C of 5min, 94 DEG C of 30s, 53 DEG C of 30s, 72 DEG C of 90s, 30 circulations, 72 DEG C of extension 10min.
Electrophoresis is verified correct gene knockout homology arm (mgdh1, mgdh2) amplified production to be connected to by homologous recombination
It is then that resistance marker Kan amplified production is homologous to gene knockout by digestion connection (Kpn I/Xho I) in pUC19 carrier
Between arm, it will finally contain plasmid pUC-2 and the suicide of gene knockout homology arm (mgdh1, mgdh2) and resistance marker (Kan)
It is recycled after the identical restriction enzyme BamH I/Hind III digestion of plasmid pK18mobSacB, it can be at through connection conversion
Function constructs gene knockout plasmid pK18-gdh.Digestion connection procedure is as shown in Figure 1.
2 electricity of embodiment turns the preparation of competence
(1) picking Acetobacter sp. single bacterium is fallen in seed culture medium, in reciprocal shaker, 30 DEG C, 100rpm item
20-24h is cultivated under part.
(2) 2% inoculum concentration is pressed, by primary seed solution switching in 50mL seed culture fluid, cultivates bacterium solution OD600Value is
Then at cooled on ice 30min between 0.3-0.8.
(3) 5min, which is centrifuged, in 4 DEG C of refrigerated centrifuges, under the conditions of 5000rpm collects thallus.
(4) 10% glycerol of pre-cooling is added, gently purges thallus, thalline were collected by centrifugation again after static 10min, and operation is such as
Step (3) is described, repeats step 3 time.
(5) 10% precooled glycerol is added after outwelling supernatant, thallus is made to suspend again, packing to new 1.5mL from
In heart pipe, every pipe is about 100-200 μ L.
The electroporated Acetobacter sp. electricity of 3 gene knockout plasmid of embodiment turns competence
(1) 10 μ L high concentration gene knockout plasmids are drawn, electricity is added and turns in competence, gently pressure-vaccum is cold on ice after mixing
But 10min.
(2) the above-mentioned competent cell for being mixed with plasmid is drawn rapidly and is added in the electric revolving cup of pre-cooling and shocked by electricity,
Voltage is set as 2500V, and the electric shock time is set as 5ms.
(3) seed culture is drawn after shocking by electricity rapidly to be based in electric revolving cup, gently whole bacterium solutions are transferred to by pressure-vaccum after mixing
In new centrifuge tube, in reciprocal shaker, 30 DEG C, cultivate 6h under the conditions of 100rpm, then draws 200 μ L and be transferred to nonreactive kind
20h or so is cultivated in sub- culture medium, then draws 200 μ L and is forwarded in nonreactive seed culture medium and cultivate 20h, then uses physiological saline
Gradient dilution is applied in resistant panel containing kanamycin, cultivates 2-3d.
(4) bacterium colony PCR verifying is carried out using primer GDH-F1, GDH-R1, correct single colonie is verified to PCR and carries out liquid
Culture is verified after extracting genomic DNA using primer GDH-F1, GDH-R1 further progress PCR, and verifying correctly illustrates gdh base
Because missing bacterium constructs successfully.
Suicide plasmid is using homologous recombination twice by gene knockout, and homologous recombination process is as shown in Fig. 2, same for the first time twice
After the recombination of source, suicide vector plasmid integration to genome；After second of homologous recombination, suicide vector plasmid is detached from genome, figure
Middle black segment is genetic fragment after knocking out.
Comparison electrophoresis is as shown in Figure 3 before and after gdh gene knockout.
Growing state of the 4. acetobacter engineering bacteria of embodiment in most suitable catalytic performance culture medium
By shake flask fermentation, 30 DEG C, 200rpm, it is incubated at most suitable catalytic performance culture medium, ferment 72h, during which not timing
Sampling measures OD by spectrophotometric600And pH is detected, and see Fig. 4, after knocking out, missing bacteria culture fluid pH no longer declines rapidly,
It ferments after 72h, the pH value of acetobacter engineering bacteria is 5.84, and the pH value of wild type acetobacter is 3.25, OD600It is worth wilder
Raw bacterium improves 1.72 times.
The host of acetobacter is changed to Acetobacter indonesiensis by comparative example 1, remaining condition and embodiment
In it is consistent
Another acetobacter engineering bacteria will be obtained after the gdh gene knockout of Acetobacter indonesiensis
A.indonesiensis△gdh。
Most suitable urge is incubated at by engineering bacteria A.indonesiensis △ gdh at 30 DEG C, 200rpm by shake flask fermentation
Change performance culture medium, ferment 72h, and not timing sampling measures OD by spectrophotometric600And pH is detected, and see Fig. 5, after knocking out,
Missing bacteria culture fluid pH no longer declines rapidly, and after the 36h that ferments, the pH value of acetobacter engineering bacteria is 5.45, and wild type acetic acid
The pH value of bacillus is 3.42, but OD600Value has dropped 30% or more compared with wild mushroom.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>a kind of acetobacter engineering bacteria and its application
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 2412
<212> DNA
<213> Acetobacter sp. CGMCC 8142
<400> 1
atgacgccac cttcttcccg gtccggggtg tttcttaacg tgatcatcgc gatctttgtt 60
ctgctagggt tgtacctggc gggcggtggc gcatggctgg cgatgcttgg cgggtcatgg 120
ttctatctgt tcagcggcat tgttctgctg gttgttgccg tcatgctgct gaaacgctgg 180
cgcgaggccc tgtgggtgta tgcagccctt gtgtttgcaa cactgatctg ggccgtcgcg 240
gaatccgggt ttgatttctg ggcgttggca ccgcgtggtg acgttattct gccccttggc 300
ttgctgcttt tgctgcccgc agtgtcacgt aatcttttgc ctgcaaaatc ccttaacagc 360
ctgccgctgg cagccgggat tgttgcaggg ctgggtgttt atggctttgc cctgacacaa 420
gacccgcagg atgttgctgg tacgttgcca agtgtagcgc agcagggtgc cccggtaaca 480
ccgggggatg tggcagaagc cgcagctggc gactggcccg cttatgggcg cacccagttt 540
ggcaaccgct attcaccgct ggcgcagatt agcccggcta acgtgaagaa cctgaaggtg 600
gcatgggttt tccgtaccca tgacctgaaa accccaaatg accccggcga aattactgac 660
gaagtcacgc caatcaaaat tcgggatacc ctctatcttt gctcgccgca ccaaatcgtc 720
tttgcgcttg atgctgcaac aggcaaggaa aagtggaagt atgacccaaa gctgaaatac 780
aaccccagct ttcagcacat gacctgccgt ggtgtttcct attctgaagc gcacgaaggc 840
gacgtaacgg ctgatggttc tcctgtgccg acagatgatt gcgcacgccg tatttttctg 900
cccaccaatg atggtcgcct gatcgcgctg aatgcggaga caggggaagc ctgtcacgct 960
tttgggaaag aaggcgcggt tgatctgtct gacggaatgc ccatgcgcga gcttgggttt 1020
tacgaaccga catcgccccc cgttgttacc ggcaaggttg tgattgtatc gggcgcggtg 1080
acagataact attccacgca cgaaccatca ggtgttacgc gggcgtatga cctgtatacg 1140
ggccgtctgg tctgggcttt tgatacggga aatcccaacc cgaacgaact gccatctgac 1200
acgcaccatt atgtgcccaa ttctcccaat tcatggatta cgtcagctta cgatgccaaa 1260
ctgaacctga tttatatccc caccggtgtt gcaacgcctg atatctgggg tggccaccgg 1320
tcggaagatc aggaacgcta tgcatccggt attctggccc tgaatgccga cacgggtgaa 1380
aaagtctggt tctatcagac tgtgcatcat gatctgtggg atatggatgt tccatcccag 1440
cccagcctcg tggatatccg ccagaaggat gggtctgttg tgcctgcgct gtatgcgcca 1500
gccaaaaccg gcaacatctt tgtgcttgat cggcgggatg ggcatctgat tgtgcctgcg 1560
ccagaaacac cggtaccgca gggggccgca ccgggcgacc atgtggtcgc aacgcagccg 1620
tattctcagt tgacgttccg ccccaagaaa aacctgacag atgcggatat gtggggtggc 1680
accatgcttg accagatggt gtgccgaatc atgttcaaga aaatgcggta cgaggggcca 1740
ttcacgccac cgtccttgca gggcacattg gtgttcccgg gtaatctggg catgtttgaa 1800
tggggtggtc tggctgtaga ccccgtgcgt caggtggcgt ttgctaaccc gattgccatt 1860
ccgtttgttt ccaagctgat tccacgcggt cccggcaacc ctgcgcagcc agacccaaaa 1920
gctggtcctt ccgggtcaga aagcggtgtg cagccgcaat acggcacacc atatggggtt 1980
gacctgcatg ccttcctgtc accgctaggt attccgtgca aacagccggg ctggggctat 2040
gttgccggta ttgacctgaa aaccaaccgg attatgtgga tgcaccgtaa cggcaccgtg 2100
cgtgatagtt caccgctgcc attgccattc aaggttggta ttccgtctct gggtgggccg 2160
ctgacaacag caggtggtgt ggccttcctg acatctacgg ctgattatta tatccgggcc 2220
tatgatgtga cgacaggcaa gcaactgtgg caggaccgtc tgcctgctgg tggtcagtcc 2280
accccaatga cgtatgaagc caacggccgc cagtttgtgg tgacggttga tggcgggcat 2340
gggtcttttg gcaccaagct gggagattac attgtggcgt atgctctgcc agacggtgca 2400
accaagcagt aa 2412
<210> 2
<211> 1495
<212> DNA
<213> Acetobacter sp. CGMCC 8142
<400> 2
agcagcttca cgcatcaagt cgcgactgac cgaaaaaggc gaacacggag ccaccactac 60
gcgctgcatc gccagaggcg cggggtcatg gaaggcttcg ataacccgtt gcgtgtcctt 120
cagaatagca gtttcctgct ctacaacact atcggggggc aggccgcctt tgctttctcc 180
aacactcatg gagcctcggg ctgcgtgaaa ccgcatcccc atttcggttg cggcttcaat 240
ctggtcgtcc agcctgatgc cattggggaa cagataaaga tgatcactgg ttgtcgtgca 300
gccagaccac agcagttccg tcatggctgt gcaggttgaa acacggatca tttctggcgt 360
caggcccgcc cagataggat agagggcctg taaccacccg aataaagagg aatcctgtgc 420
cgcagggata acgcgcgtca gggtctggta catatggtgg tgtgtattca ccatgccggg 480
catgacaacg tggcccgtca tatccagcac ctcatctgcg tcgggcaggg gagtgtctgc 540
tgtgccaatg gccgcaacct gtcggccttt taccagtacc cagcctcctg ccagttcgtc 600
acgatggtcg ttcatggtta ccagacgcag ggcatttttc agcagaaggg tgcgcatcgg 660
tctctctttg acacgtcagg gaaagaataa ggtgtttggc tattctaaca ggtcgctcgg 720
gtaacggaaa acccgtgtgg aacagtcagc aagtttcgac gtttgggctc attgcggtct 780
gtccatttat ttgatttccg aaagaaaaaa gaagccttga tcaaaccgga acatccggcg 840
caaccagcct tgtctgactg atagcgcctg aattttccgg gacaatcatg cggtttcctt 900
ataaagtgga ccatccttgt cgatatttct gtctaacagc cagggttcat ttgcatgacg 960
ccaccttctt cccggtccgg ggtgtttctt aacgtgatca tcgcgatctt tgttctgctg 1020
gggctgtacc tggcgggcgg tggcgcatgg ctggcgatgc ttggcgggtc atggttctat 1080
ctgttcagcg gtattgttct gcttgttgtg gccgtcatgc tgctgaaacg ctggcgcgag 1140
gccctgtggg tgtatgcagc tcttgtgttt gcaacgctcg tctgggcagt cgcggaatcc 1200
gggtttgatt tctgggcgtt ggcaccgcgt ggtgacgtga ttctgcccct tggcctgttg 1260
cttttgctgc ctgtggtgtc acgtgctctt ttgcccgcaa aaccccttaa cagcctgccg 1320
ctggcagccg ggattgttgc agggctgggt gtttatggct ttgccctgac gcaagacccg 1380
caggatgttt ctggtacgtt gccaagtgta gcgcagcagg gtgccccggt aacaccgggg 1440
gatgtggcag aagccgcagc gggcgactgg cctgcttatg ggcgcacgca gtttg 1495
<210> 3
<211> 1515
<212> DNA
<213> Acetobacter sp. CGMCC 8142
<400> 3
ctaacccgat tgccattccg tttgtttcca aactgatccc acgcggtccc ggcaaccctg 60
cgcagccaga cccaaaagct ggtccttccg ggtcagaaag tggtgtgcag ccgcaatacg 120
gcacaccata tggggttgac ctgcatgcct tcctgtcacc gctgggtatt ccgtgcaaac 180
agccgggctg gggctatgtg gccggtattg acctgaaaac caaccggatt atgtggatgc 240
accgtaacgg caccgtgcgt gatagttcac cgctgccatt gccattcaag gttggtattc 300
cgtctctggg tgggccgctg acaacagcag gtggtgtggc cttcctgaca tctacggctg 360
attattatat ccgggcctat gatgtgacga caggcaagca actgtggcag gaccgtctgc 420
ctgcgggtgg tcagtccacc ccaatgacgt atgaagccaa cggccgccag tttgtggtga 480
cggttgatgg cgggcatggg tcttttggca ccaagctggg tgattacatt gtggcgtatg 540
ctctgccaga cggtgcaacc aagcagtaat actcagtcat ttttaatgtc ggggcgagta 600
gggaaacctg ctcgcctttt tttatttcgg cgtaatgttt gcgtggtgga aggacaaagg 660
aatgtatgtg gttcttggtg caacagggca tgtgggcagt attgttgcgg atcagttgct 720
ggacgcccag tgccccgtaa ctattgtgac ccgtagcaga gaaaaagcgg cgctatggca 780
gcggaaagga gccgtagccg cagtggcggg tatttacgac cccgctgcgc ttgaagatgt 840
ttttcgcaaa gcatcgcgcg tttttctgct caacccgcct gcaccggtat cggaagatac 900
cgatacggtt gagcgccagt cggtccatgc cattctggct gcattaaagg gcgttcagct 960
tgaaaaaatt gttgttcaat caacatacgg cgcacaacct ggcccgcatt gtggggatct 1020
gggtgttctg tatgaacttg aggaaggtgt gcggagtcta ggcgtgccaa gctgctcttt 1080
gcgggcggct tattacatga gcaactgggt ttcttccgca aaacaggtgc gtgagacagg 1140
agaattttcc acactattgc ccgtcgggct gaaagtgccg atggttgcac cacaggatgt 1200
cggggcgctg gccgcaagac tgctgatgtc agaggtcaca gaaacaggtc tttatgcgtg 1260
tgaagggcca agcctgtatg cgccgcagga tgttgccaca gttcttgggc gcgtagtggg 1320
tcgccccgta tcggtccgtg aaatcccgtc ggctgactgg ctggcttatt atcgtgaaaa 1380
cggtttttcc gaacaggcag cgcagtctta tgcacacatg accaaactgt tcataagcca 1440
gccccacgaa caggcaggga tacgttgcaa gggttcaaca gggctggagg cgtattttaa 1500
gggcgtgttt ggggc 1515
Claims (10)
1. a kind of acetobacter engineering bacteria, which is characterized in that be the base in acetobacter Acetobacter sp.CGMCC 8142
Because having knocked out glucose dehydrogenase gene in group.
2. acetobacter engineering bacteria as described in claim 1, which is characterized in that the nucleotide of the glucose dehydrogenase gene
Sequence is as shown in SEQ ID NO.1.
3. the construction method of acetobacter engineering bacteria of any of claims 1 or 2, which is characterized in that the described method includes:
(1) gene knockout plasmid is constructed；
(2) gene knockout plasmid is converted to Acetobacter sp competent cell.
4. according to the method described in claim 3, it is characterized in that, the gene knockout plasmid is with pK18mobSacB for load
Body expresses gene shown in gene shown in the NO.2 of ID containing SEQ and the NO.3 of ID containing SEQ.
5. application of the acetobacter engineering bacteria of any of claims 1 or 2 in catalysis biochemical reaction.
6. acetobacter engineering bacteria of any of claims 1 or 2 is in production 3- hydracrylic acid, acrylic acid, 1,3-PD, the third two
Application in sour or poly- 3- hydracrylic acid.
7. acetobacter engineering bacteria of any of claims 1 or 2 is in the application of food or field of fodder.
8. acetobacter engineering bacteria of any of claims 1 or 2 is in terms of the additive or preservative that prepare food or feed
Using.
9. acetobacter engineering bacteria of any of claims 1 or 2 is in the application of chemical industry or pharmaceutical field.
10. acetobacter engineering bacteria of any of claims 1 or 2 preparation biodegradable thermoplastic's polyester, insecticide, coating,
Application in terms of adhesive, plasticizer or emulsifier.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811375476.8A CN109370972A (en) | 2018-11-19 | 2018-11-19 | A kind of acetobacter engineering bacteria and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811375476.8A CN109370972A (en) | 2018-11-19 | 2018-11-19 | A kind of acetobacter engineering bacteria and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109370972A true CN109370972A (en) | 2019-02-22 |
Family
ID=65389255
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811375476.8A Pending CN109370972A (en) | 2018-11-19 | 2018-11-19 | A kind of acetobacter engineering bacteria and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109370972A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112852857A (en) * | 2021-02-06 | 2021-05-28 | 江南大学 | Method for knocking out multiple genes in gluconobacter oxydans |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003339392A (en) * | 2002-05-24 | 2003-12-02 | Kumamoto Technology & Industry Foundation | Method for producing bacterial cellulose |
| CN102146415A (en) * | 2010-07-16 | 2011-08-10 | 华东理工大学 | Gene knockout bacterium of gluconobacter oxydans and preparation method thereof |
| CN102392056A (en) * | 2011-12-09 | 2012-03-28 | 华东理工大学 | Genetically engineered strain and method for producing dihydroxyacetone by using the same |
| CN103525727A (en) * | 2013-10-10 | 2014-01-22 | 江南大学 | Method for transforming 1,3-propylene glycol into 3-hydroxypropionic acid by applying resting cells |
| CN104745645A (en) * | 2015-02-11 | 2015-07-01 | 江南大学 | Method for synthesizing acrylic acid through biotransformation of acraldehyde |
| CN105483166A (en) * | 2014-10-09 | 2016-04-13 | 天水绿健生物技术有限责任公司 | Biological preparation method of succinic acid |
-
2018
- 2018-11-19 CN CN201811375476.8A patent/CN109370972A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003339392A (en) * | 2002-05-24 | 2003-12-02 | Kumamoto Technology & Industry Foundation | Method for producing bacterial cellulose |
| CN102146415A (en) * | 2010-07-16 | 2011-08-10 | 华东理工大学 | Gene knockout bacterium of gluconobacter oxydans and preparation method thereof |
| CN102392056A (en) * | 2011-12-09 | 2012-03-28 | 华东理工大学 | Genetically engineered strain and method for producing dihydroxyacetone by using the same |
| CN103525727A (en) * | 2013-10-10 | 2014-01-22 | 江南大学 | Method for transforming 1,3-propylene glycol into 3-hydroxypropionic acid by applying resting cells |
| CN105483166A (en) * | 2014-10-09 | 2016-04-13 | 天水绿健生物技术有限责任公司 | Biological preparation method of succinic acid |
| CN104745645A (en) * | 2015-02-11 | 2015-07-01 | 江南大学 | Method for synthesizing acrylic acid through biotransformation of acraldehyde |
Non-Patent Citations (2)
| Title |
|---|
| JUN LI ET AL.: ""Immobilization of Acetobacter sp. CGMCC 8142 for Efficient Biocatalysis of 1,3-propanediol to 3-hydroxypropionic Acid"", 《BIOTECHNOLOGY AND BIOPROCESS ENGINEERING》 * |
| 祝坤: ""氧化葡萄糖酸杆菌对葡萄糖利用的代谢改造及催化研究"", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112852857A (en) * | 2021-02-06 | 2021-05-28 | 江南大学 | Method for knocking out multiple genes in gluconobacter oxydans |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| BRPI0618074A2 (en) | thermophilic organisms for converting lignocellulosic biomass to ethanol | |
| CN1225125A (en) | Stable recombinant yeasts for fermenting xylose to ethanol | |
| CN107709540A (en) | Novel warehouse Delhi A Ziweishi Pichia pastoris strains NG7 and application thereof | |
| CN110157654B (en) | Bacillus natto recombinant strain and construction method and application thereof | |
| CN102373230A (en) | Nucleotide sequence of Clostridium D-tagatose 3-epimerase and application thereof | |
| CN105420154A (en) | Double knockout recombinant rhodococcus as well as construction method and application thereof | |
| CN101565710B (en) | 3-sterone-Delta[1]-dehydrogenase gene, relevant carriers, engineering strains and applications thereof | |
| CN101565709A (en) | 3-sterone-9Alpha-hydroxylation enzyme gene, 3-sterone-9Alpha-hydroxylation enzyme reductase gene, relevant carriers, engineering bacteria and applications thereof | |
| CN104593407B (en) | Pichia stipitis gene expression system and its structure and application | |
| CN104789586A (en) | Escherichia coli genome integration vector, genetically engineered bacterium and application of genetically engineered bacterium to xylitol production | |
| CN111154705B (en) | Bacillus thermoglucosidasius engineering bacterium and construction method and application thereof | |
| CN109370972A (en) | A kind of acetobacter engineering bacteria and its application | |
| CN110004070B (en) | Xylanase-producing Aspergillus niger genetically engineered bacterium and construction method and application thereof | |
| CN104974945B (en) | A kind of saccharomyces cerevisiae of overexpression MIG1 genes and preparation method and application | |
| CN114107356B (en) | Method for transforming pseudomonas putida to assimilate D-galactose | |
| CN104561081B (en) | One plant can utilize the expression system of the yeast Candida amazonensis of xylose | |
| CN101525580B (en) | Amphiploid histidine auxotroph saccharomyces cerevisiae and constructing method thereof | |
| CN101892228B (en) | Engineering bacteria with high tolerance to acrylamide and acrylonitrile for producing nitrile hydratase and application thereof | |
| CN107083375B (en) | Medium-temperature alpha-amylase and gene and application thereof | |
| CN105349567A (en) | Signal peptide and application thereof in konjac powder production of L-ornithine recombinant bacteria | |
| CN104962574B (en) | A kind of arthrobacterium expression plasmid and application | |
| CN105838730A (en) | Engineered bacteria for phytosterol conversion and construction method and application of engineered bacteria | |
| CN104630257B (en) | One plant can utilize the expression system of the yeast Spathaspora passalidarum of xylose | |
| CN110713940B (en) | High-yield heavy oil aureobasidium pullulans strain and construction method and application thereof | |
| CN103509817A (en) | Construction method and application of high-yield oil and linoleic acid genetically-engineered bacterium of Rhodotorula glutinis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190222 |
|
| RJ01 | Rejection of invention patent application after publication |