CN108531434A - A method of improving Raoul bacterium 2,5- furandicarboxylic acid yield - Google Patents
A method of improving Raoul bacterium 2,5- furandicarboxylic acid yield Download PDFInfo
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Abstract
The invention discloses a kind of methods of 2,5 furandicarboxylic acid yield of raising Raoul bacterium, belong to metabolic engineering technical field.The present invention has knocked out five 5 hydroxymethylfurfural reductase genes in the Raoul bacterium for producing 2,5 furandicarboxylic acids simultaneously using the method for homologous recombination.The Raoul bacterium after five genes is knocked out during biotransformation method produces 2,5 furandicarboxylic acids, 2,5 furandicarboxylic acid contents reduce 37.0% than starting strain in conversion fluid, and the content of 2,5 furandicarboxylic acids increases 23.2%.
Description
Technical field
The present invention relates to a kind of methods improving Raoul bacterium 2,5-furandicarboxylic acid yield, belong to metabolic engineering technology neck
Domain.
Background technology
2,5- furandicarboxylic acids (2,5-Furandicarboxylic acid, FDCA) be a kind of important industrial chemicals and
The precursor of organic synthesis can be used to prepare a variety of lipid furans or alkyl-substituted derivative, and is classified as most by U.S. Department of Energy
One of promising 12 kinds of platform chemicals.In addition, the substitute that 2,5-furandicarboxylic acid is also used as terephthalic acid (TPA) is used
It manufactures the plastic cement products of polyesters, has broad application prospects.
Currently, the method for preparing 2,5-furandicarboxylic acid mainly has chemical synthesis and biotransformation method.Chemical synthesis
Generally using noble metal as catalyst, the conditions such as high pressure, high temperature and organic solvent are needed, to cause higher manufacturing cost
With Heavy environmental pollution problem.Biotransformation method is given birth to generally using 5 hydroxymethyl furfural as substrate using enzyme or resting cell method
At 2,5- furandicarboxylic acids.Compared with chemical synthesis, biotransformation method has many advantages, such as mild condition, low toxicity and environmental protection.So
The compound and toxic action due to 5 hydroxymethyl furfural to cell, the only microorganism of only a few can degrade.Wherein, it solves
Ornithine Raoul bacterium (Raoultella ornithinolytica BF60) can be catalyzed 5 hydroxymethyl furfural and generate 2,5- furans
It mutters dioctyl phthalate, there are the advantages such as higher substrate conversion efficiency and application prospect.But the by-product 2,5- furans in catalytic process
The generation of dimethanol limits efficiently synthesizing for 2,5- furandicarboxylic acids.
During solving the catalysis 5 hydroxymethyl furfural oxidation of ornithine Raoul bacterium, 5 hydroxymethyl furfural can be gone back first
Originally it was 2,5-FDM, 2,5-furandicarboxylic acid is then generated by gradually oxidation again.The study found that in Escherichia coli or ferment
The enzyme that 5 hydroxymethyl furfural reduction is catalyzed in mother is a variety of non-specific oxidation reductases of intracellular.In solution ornithine Raoul bacterium
In there is also similar oxidoreducing enzyme, 5 hydroxymethyl furfural is reduced to 2,5-FDM.Therefore, this research is by striking
Except five 5 hydroxymethyl furfural reductase genes in solution ornithine Raoul bacterium, the life of by-product 2,5-FDM is reduced
At to improve the yield of 2,5-furandicarboxylic acid.
Invention content
The purpose of the present invention is by knocking out five 5 hydroxymethyl furfural reductase genes (akR, aldR, AdhP1, AdhP2
And dkgA), the generation of by-product 2,5-FDM is reduced, and then improve the yield of 2,5-furandicarboxylic acid.Invention content
Various bacterial strains simple to operation, can belonging to extensively using Raoultella.
The first purpose of the invention is to provide a kind of solution ornithine Raoul bacterium of 2,5-furandicarboxylic acid output increased,
Be with solve ornithine Raoul bacterium (Raoultella ornithinolytica) BF60 (bacterial strain in paper Hossain,
G.S.,Yuan,H.,Li,J.,Shin,H.D.,Wang,M.,Du,G.,Chen,J.,Liu,L.2017.Metabolic
Engineering of Raoultella ornithinolytica BF60for Production of 2,5-
Furandicarboxylic Acid from 5-Hydroxymethylfurfural.Appl Environ Microbiol,83
(1), disclosed in e02312-16.) it is starting strain, knock out at least three base in akR, aldR, AdhP1, AdhP2 and dkgA
Cause.
In one embodiment of the invention, described knock out is knocked out with Red recombination systems.
In one embodiment of the invention, the knockout is as follows：
(1) it using pKD13 plasmids as template, separately designs containing target gene akR, aldR, AdhP1, AdhP2 or dkgA two
The primer amplification of side homologous fragment goes out to knock out the target practice segment of target gene；
(2) the target practice segment electrotransformation obtained in (1) is entered into the pKD46 plasmids containing chloramphenicol resistance gene
In R.ornithinolytica BF60, it is coated with the tablet containing kalamycin resistance, positive gram is obtained by bacterium colony PCR verifications
Grand bacterial strain.
(3) positive colony bacterium obtained in high-temperature cultivation (2), loses temperature sensitive type pKD46 plasmids, and then electrotransformation enters
PCP20 plasmids, high-temperature cultivation eliminate kalamycin resistance gene label, are then filtered out while being lost using different resistant panels
Fall the mutant strain of pCP20 plasmids and kalamycin resistance gene label, this mutant strain is what target gene lacked
R.ornithinolytica。
(4) step (1)~(3) are repeated, with knock out five 5 hydroxymethyl furfural reductase genes (akR, aldR, AdhP1,
AdhP2 and dkgA).
In one embodiment of the invention, in five 5 hydroxymethyl furfural reductase genes, aldehyde ketone reduction
Enzyme coding gene akR is as shown in SEQ ID NO.1, and alcohol dehydrogenase encoding gene aldR is as shown in SEQ ID NO.2, alcohol dehydrogenase
Encoding gene AdhP1 is as shown in SEQ ID NO.3, and alcohol dehydrogenase encoding gene AdhP2 is as shown in SEQ ID NO.4, and 2,5- bis-
Dehydrogenation gluconic acid reductase encoding gene dkgA is as shown in SEQ ID NO.5.
Second object of the present invention is to provide a kind of improve and solves ornithine Raoul bacterium 2,5- furandicarboxylic acid yield
Method.
In one embodiment of the invention, the method is as follows：
(1) it using pKD13 plasmids as template, separately designs containing target gene akR, aldR, AdhP1, AdhP2 or dkgA two
The primer of side homologous fragment amplifies the target practice segment that need to knock out target gene；
(2) the target practice segment electrotransformation obtained in (1) is entered into the pKD46 plasmids containing chloramphenicol resistance gene
In R.ornithinolytica BF60, it is coated with the tablet containing kalamycin resistance, positive gram is obtained by bacterium colony PCR verifications
Grand bacterial strain；
(3) positive colony bacterium obtained in high-temperature cultivation (2), loses temperature sensitive type pKD46 plasmids, and then electrotransformation enters
PCP20 plasmids, high-temperature cultivation eliminate kalamycin resistance gene label, are then filtered out while being lost using different resistant panels
Fall the mutant strain of pCP20 plasmids and kalamycin resistance gene label, this mutant strain is what target gene lacked
R.ornithinolytica；
(4) step (1)~(3) are repeated, with knock out five 5 hydroxymethyl furfural reductase genes (akR, aldR, AdhP1,
AdhP2 and dkgA).
Third object of the present invention is to provide the application recombination R.ornithinolytica BF60 to produce 2,5- furans
It mutters the method for dioctyl phthalate.
In one embodiment of the invention, the method is quiet with the recombination R.ornithinolytica BF60
Breath cell is biocatalyst, and using 5 hydroxymethyl furfural as substrate, catalytic production 2,5-furandicarboxylic acid is carried out in conical flask.
In one embodiment of the invention, the recombination R.ornithinolytica BF60 are thin according to 1.5~5g
The dosage of born of the same parents/g 5 hydroxymethyl furfurals carries out catalysis reaction.
In one embodiment of the invention, the catalysis is 12~60h of reaction at 20~37 DEG C.
In one embodiment of the invention, the recombination R.ornithinolytica BF60 are cultivated in the steps below
It obtains：
(1) seed liquor culture：It is inoculated in liquid LB from tablet picking recombination R.ornithinolytica BF60 single bacterium colonies
In culture medium (tryptone 10g/L, yeast powder 5g/L, NaCl 10g/L), 30 DEG C, 220rpm cultures 12h；
(2) thalline culture：Seed liquor is transferred to TB culture mediums (tryptone 12g/L, ferment with the inoculum concentration of 1% (V/V)
Female powder 24g/L, K2HPO4 12.5g/L、KH2PO42.3g/L, glycerine 4mL/L) in, 30 DEG C, 220rpm culture for 24 hours.
The present invention also provides the applications for recombinating R.ornithinolytica BF60 in food, biology, chemical field.
Advantageous effect：The present invention has knocked out five 5- methylol chaffs in Raoul bacterium by the method for homologous recombination simultaneously
Aldehyde reductase gene (akR, aldR, AdhP1, AdhP2 and dkgA), weakens host strain and is reduced to 2,5- by 5 hydroxymethyl furfural
The approach of furyl dimethyl carbinol promotes 5 hydroxymethyl furfural and is converted into 2 to reduce the generation of 2,5-FDM,
5- furandicarboxylic acids.During embodiment living things catalysis, while knocking out akR, aldR, AdhP1, AdhP2 and dkgA gene
The content of 2,5-FDM reduces 37.0%, while the content of 2,5-furandicarboxylic acid than starting strain in Raoul bacterium
Increase 23.2%.
Description of the drawings
Fig. 1 gene knockout mutant strains PCR verifies electrophoretogram；Wherein, M：Marker；1、3、5、7：Respectively aldR-dkgA,
The original gene of akR, AdhP1 and AdhP2；2：The segment that aldR-dkgA genes are lacked and are inserted into behind the sites FRT simultaneously；4：akR
Gene delection and the segment being inserted into behind the sites FRT；6：AdhP1 gene delections and the segment being inserted into behind the sites FRT；8：AdhP2 bases
Because lacking and being inserted into the segment behind the sites FRT.
The missing of Fig. 2 difference 5 hydroxymethyl furfural genes is to the bacterial strain biology after transformation R.ornithinolytica BF60
The influence of catalytic efficiency；" Δ " indicates to knock out.
Specific implementation mode
The following examples are further illustrations of the invention, rather than limiting the invention.
The assay method of 2,5- furyl dimethyl carbinols：High performance liquid chromatography (HPLC) detection method：Agilent 1260, VWD are examined
Survey device, Detection wavelength 230nm, YMC-Pack ODS-A chromatographic columns, 35 DEG C, flow velocity 0.6ml/min of column temperature, mobile phase：(A) pure
Water, (B) acetonitrile；Gradient elution program：0min, 5%B；15min, 100%B；20min, 5%B；30min, 5%B.
The assay method of 2,5- furandicarboxylic acids：High performance liquid chromatography (HPLC) detection method：Agilent 1260, VWD are examined
Survey device, Detection wavelength 268nm, Aminex HPX-87H chromatographic columns, 60 DEG C, flow velocity 0.6ml/min, mobile phase 10mM of column temperature
H2SO4。
Embodiment 1 using Red recombination systems this example demonstrates that knock out akR genes in R.ornithinolytica BF60
Method.
1) from the monoclonal inoculation of picking R.ornithinolytica BF60 on tablet in LB liquid medium,
30 DEG C, 220rpm cultivates to OD600When=0.6, electrotransformation competence is prepared.
2) the pKD46 plasmid electrotransformations containing chloramphenicol resistance marker are entered in the 1) competence described in step, is coated with
Chlorampenicol resistant tablet is simultaneously put into 30 DEG C of incubator culture 12h.
3) picking 2) single bacterium colony in step tablet, in access LB liquid medium (chlorampenicol resistant), 30 DEG C, 220rpm
It cultivates to OD600When=0.2, L-arabinose (final concentration 10mM) is added and continues culture to OD600=0.6, electricity is prepared again to be turned
Change competence.
4) using pKD13 plasmids as template, primer amplification of the design both ends with akR genes both ends homologous fragment goes out containing card
The linear DNA target practice segment of that mycin resistant gene label.Primer sequence is：
Sense primer akR-D-F:GCCAGGCGCTAAAAAATCTCAAGATCCCGCGCGAAAACGTGGTGGTCGCCACC
AAGGTGTTCGGTGAATTCTCCGTGGACCTGCAG
Downstream primer akR-D-R:CTGATGGAGCAGCCACGCGAGCGCGACCTGCGCGACCGTCGCGCCTTTGGCGT
CCGCCACCTCGCGCGGTACCGAGCTCGGATCCG
PCR amplification, amplification reaction condition are carried out by template of pKD13：95 DEG C of pre-degeneration 3min；95 DEG C denaturation 30s, 55 DEG C
15s, 72 DEG C of 2min are recycled 31 times；72℃5min；12 DEG C of heat preservations.PCR product is cut after agarose gel electrophoresis detaches
Glue recycles, and obtains the target practice segment of akR genes.
5) the target practice segment electrotransformation that step 4) obtains is entered in the competent cell in step 3), coating kanamycins is anti-
Property LB tablets, 30 DEG C culture 12h.Then the single bacterium colony on picking tablet carries out bacterium colony PCR verifications, screening positive clone.
6) (40 DEG C) cultures of the positive colony high temperature in picking step 5), lose temperature sensitive type pKD46 plasmids, then electric again
PCP20 plasmids are transformed into, picking positive colony bacterium, (40 DEG C) cultures of access LB liquid medium high temperature are with induced expression FLP weights
The expression of group enzyme, to eliminate the kalamycin resistance gene being introduced into step 5) in genome label.
7) with R.ornithinolytica BF60 starting strains and R.ornithinolytica BF60_ Δ akR genes
Group is that template carries out PCR amplification verification, and the results are shown in Figure 1.PCR items shown in PCR bands 1038bp and swimming lane 4 shown in swimming lane 3
Band 640bp is consistent with theory, illustrates that akR gene deletion strains are built successfully.
AldR, AdhP1, AdhP2 and dkgA are knocked out according to above-mentioned identical strategy, wherein for expanding target practice sequence
The primer of row is respectively：AldR-D-F：TCGTCATCTGGCCGGGATATCTTCTCCGCCGGCAGTTTTCTCTTATTGAGGTG
ATGAGCAGAATTCTCCGTGGACCTGCAG；AldR-D-R：TTAAACTCCTCTCCTTCGCATCCGATGACGCGAAGGAGA
GGGCGTATCAGCAGGGGAGAAGGTACCGAGCTCGGATCCG；AdhP1-D-F：
AAATCATTGGCCGCGTCGCGGCGCTGGGCAGCGCGGCGCAGGAGAAAGGGCTGAAGGTCGGAATTCTCCGTGGACCT
GCAG；AdhP1-D-R：CGGATCAGGGTAAAGGCGGGTACCGGGAACGGCTTCATCACCGCGCCGACGGTATGGAATGG
TACCGAGCTCGGATCCG；AdhP2-D-F：CCCTGCGTGCCGGGGCATGAAATCGTCGGCCGGGTGACCGCCGTCGGCG
AACACGTCTCCGAATTCTCCGTGGACCTGCAG；AdhP2-D-R：CGCCAATCATCGACCCGGCAATCGACCGACGACG
AAATATCAGGTTAAACACTTCCGGCGGGTACCGAGCTCGGATCCG；DkgA-D-F：
ATGACACATCCAACCGTGATAAAACTGCACGACGGCAACCTGATGCCACAGCTGGGCCTCGGCGTGAATTCTCCGTG
GACCTGCAG；DkgA-D-R：TTAGCCGCCAAACTGGTCAGGATCTGGCCCGAGGCGCTTGTTGATATCCAGCTTCGCC
ATTGCTCGGTACCGAGCTCGGATCCG.The electrophoretogram of gene knock-out bacterial strain is as shown in Figure 1, PCR bands shown in swimming lane 1
PCR bands 350bp is consistent with theory shown in 2364bp and swimming lane 2, illustrates aldR and dkgA genes while the strain construction of missing
Success；PCR bands 602bp shown in PCR bands 1020bp and swimming lane 6 shown in swimming lane 5 is consistent with theory, illustrates that AdhP1 genes lack
Lose strain construction success；PCR bands 554bp shown in PCR bands 1050bp and swimming lane 8 shown in swimming lane 7 is consistent with theory, explanation
AdhP2 gene deletion strains are built successfully.
The application of embodiment 2 while the recombination R.ornithinolytica BF60- for knocking out 55 hydroxymethyl furfural reductases
Xiv carries out the production of 2,5- furandicarboxylic acids.
Seed liquor culture：It is inoculated in LB liquid medium from the single bacterium colony of tablet picking BF60-xiv bacterial strains, 30 DEG C,
220rpm cultivates 12h.
Thalline culture：Seed liquor is transferred to the inoculum concentration of 1% (V/V) in TB culture mediums, 30 DEG C, 220rpm cultures
24h。
Living things catalysis：(4 DEG C, 6000rpm, 10min) is collected by centrifugation in the thalline that previous step obtains, with 50mM phosphoric acid
Twice, thalline then is resuspended to OD with same buffer solution in salt buffer (pH 8.0) washing thalline600=100.Then it is added
100mM substrate 5 hydroxymethyl furfurals carry out biocatalytic reaction and produce 2,5-furandicarboxylic acid.
Sampling carries out HPLC analyses after being catalyzed 48h.Catalytic result shows the 2,5-furandicarboxylic acid production of BF60-xiv bacterial strains
Amount can reach 62.3mM, and 23.2% is improved than original strain.Meanwhile 2,5-FDM content is 25.2mM, than original
Beginning bacterial strain reduces 37.0%.
Using above-mentioned same procedure knockout is knocked out and combines to the single of akR, aldR, AdhP1, AdhP2 and dkgA gene
Recombinant bacterium carries out catalysis reaction, and the results are shown in Figure 2.It can be seen that in the mutant strain that individual gene lacks from result in figure,
Only the content of 2,5-FDM has apparent reduction, while 2,5-furandicarboxylic acid in the mutant strain of aldR gene delections
Content improve.And for the combination deletion mutation strain of multiple genes, the content of 2,5-FDM has
Certain reduction, while the content of 2,5-furandicarboxylic acid also has different degrees of raising.When 5 genes lack simultaneously, 2,
The content of 5- furyl dimethyl carbinols is minimum, while the content of 2,5-furandicarboxylic acid reaches peak.Due to
In R.ornithinolytica BF60 bacterial strains, catalysis 5 hydroxymethyl furfural be reduced to 2,5-FDM akR, aldR,
AdhP1, AdhP2 and dkgA gene are synergistic effects.Therefore, the missing of individual gene not can significantly reduce 2,5- furans two
The generation of methanol, only multiple genes can just reduce the content of 2,5-FDM when lacking simultaneously.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not limited to the present invention, any to be familiar with this skill
The people of art can do various change and modification, therefore the protection model of the present invention without departing from the spirit and scope of the present invention
Enclosing be subject to what claims were defined.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>A method of improving Raoul bacterium 2,5- furandicarboxylic acid yield
<160> 15
<170> PatentIn version 3.3
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atgcagatca acgctattgg tactaactcc gccagccagc cgctggccgc gatggccatc 60
tcccgccgcg agccggggcc acacgatgtg cagatcgcca tcgactactg cggcgtctgt 120
cactccgacc tgcaccaggc ccgctccgaa tgggccggga cgatctatcc ctgcgtgccg 180
gggcatgaaa tcgtcggccg ggtgaccgcc gtcggcgaac acgtctccgg ccacgcggtg 240
ggcgacctgg tgggcgtcgg ctgtatggtc gacagctgtc agcagtgcga agagtgcgag 300
gaagggctgg aaaactactg tgaccacacg gtgctcacct ataacggccc cactcaggat 360
gcgcccggcc atacgctggg cggctactcc cagcaaatcg tggttcacga gcgctacgtg 420
ctgcgcgtgc gccacccgca ggagcagctg gccgccgtag cgccgctgct gtgcgccggt 480
atcaccacat actcgccgct gcgccactgg aacgtcgggc cgggcaaaaa agtgggcatc 540
gtcggcatcg gcggactggg acacatgggg atcaagttgg ctcacgccat gggcgctcac 600
gtggtggcct ttaccacctc ggaatccaag cgtgacgcgg ccaaagagct gggggcggac 660
gaagtggtgg tgtcgcgcaa cgaggcggag atggcggcgc acgtcaaaag cttcgacttt 720
attcttaaca ccgtggcggc accacacaac ctcgatgcct tcaccacgct gcttaaacgc 780
gacggtaccc tgaccctggt gggcgcgccg gcaactccgc acccgtcgcc ggaagtgttt 840
aacctgatat ttcgtcgtcg gtcgattgcc gggtcgatga ttggcgggat cccggaaacc 900
caggagatgc tcgatttctg cgccgaacac ggcatcgtcg ccgatattga gctgatccgc 960
ggagatcgga tcaacgaagc gtgggaaagg atggtcaacg gcgacgtcaa atatcgcttc 1020
gtgatcgaca tcgccaccct cgccggctaa 1050
<210> 5
<211> 828
<212> DNA
<213>Artificial sequence
<400> 5
atgacacatc caaccgtgat aaaactgcac gacggcaacc tgatgccaca gctgggcctc 60
ggcgtgtgga aagcaggcaa cgaggaggtc gtctccgcca ttcacaaggc gctggaagtc 120
ggttatcgct cttttgacac cgccgccgcg taccaaaacg aagccggcgt cggcagcgcg 180
ctgagccagg ctggcgtccc tcgcgatgag ctgtttatca ccaccaagct atggaacgac 240
gatcaaaaac gtccgcatga agccctgcag gagagcctga gcaagctgca gctcgactac 300
gtcgatctgt atttaatgca ctggccggtt ccggcgatcg accattttgt tgaagcctgg 360
aagggcatga ttgagctcca aaaacagggg ctggccaaaa gcatcggcgt ctgtaatttc 420
caggttcacc acctgcagcg gctgcttgat gaaaccggcg tcgtgccggt gattaatcag 480
gtagagctcc atcccctgct ccagcagcgc cagctgcacg cctggaacgc gacacacaaa 540
atccagaccg aatcctggag cccgctggct cagggcggcg aaggcgtatt cgatcagaaa 600
atcgtccgcg agctggccga taagtacggc aaaacgccgg cgcagattgt catccgctgg 660
catctcgata acggcctggt ggtgatccct aaatcggtga ccccttcgcg tatcgtggag 720
aactttaacg tctgggactt ccgtcttgat aaagacgaac tgggagcaat ggcgaagctg 780
gatatcaaca agcgcctcgg gccagatcct gaccagtttg gcggctaa 828
<210> 6
<211> 86
<212> DNA
<213>Artificial sequence
<400> 6
gccaggcgct aaaaaatctc aagatcccgc gcgaaaacgt ggtggtcgcc accaaggtgt 60
tcggtgaatt ctccgtggac ctgcag 86
<210> 7
<211> 86
<212> DNA
<213>Artificial sequence
<400> 7
ctgatggagc agccacgcga gcgcgacctg cgcgaccgtc gcgcctttgg cgtccgccac 60
ctcgcgcggt accgagctcg gatccg 86
<210> 8
<211> 81
<212> DNA
<213>Artificial sequence
<400> 8
tcgtcatctg gccgggatat cttctccgcc ggcagttttc tcttattgag gtgatgagca 60
gaattctccg tggacctgca g 81
<210> 9
<211> 79
<212> DNA
<213>Artificial sequence
<400> 9
ttaaactcct ctccttcgca tccgatgacg cgaaggagag ggcgtatcag caggggagaa 60
ggtaccgagc tcggatccg 79
<210> 10
<211> 81
<212> DNA
<213>Artificial sequence
<400> 10
aaatcattgg ccgcgtcgcg gcgctgggca gcgcggcgca ggagaaaggg ctgaaggtcg 60
gaattctccg tggacctgca g 81
<210> 11
<211> 79
<212> DNA
<213>Artificial sequence
<400> 11
cggatcaggg taaaggcggg taccgggaac ggcttcatca ccgcgccgac ggtatggaat 60
ggtaccgagc tcggatccg 79
<210> 12
<211> 81
<212> DNA
<213>Artificial sequence
<400> 12
ccctgcgtgc cggggcatga aatcgtcggc cgggtgaccg ccgtcggcga acacgtctcc 60
gaattctccg tggacctgca g 81
<210> 13
<211> 79
<212> DNA
<213>Artificial sequence
<400> 13
cgccaatcat cgacccggca atcgaccgac gacgaaatat caggttaaac acttccggcg 60
ggtaccgagc tcggatccg 79
<210> 14
<211> 86
<212> DNA
<213>Artificial sequence
<400> 14
atgacacatc caaccgtgat aaaactgcac gacggcaacc tgatgccaca gctgggcctc 60
ggcgtgaatt ctccgtggac ctgcag 86
<210> 15
<211> 84
<212> DNA
<213>Artificial sequence
<400> 15
ttagccgcca aactggtcag gatctggccc gaggcgcttg ttgatatcca gcttcgccat 60
tgctcggtac cgagctcgga tccg 84
Claims (10)
1. a kind of recombination solution ornithine Raoul bacterium of 2,5-furandicarboxylic acid output increased, which is characterized in that solve ornithine
Raoul bacterium (Raoultella ornithinolytica) BF60 is starting strain, knocks out five 5 hydroxymethyl furfural reductases
In at least three.
2. recombination according to claim 1 solves ornithine Raoul bacterium, which is characterized in that five 5 hydroxymethyl furfurals
Reductase includes akR, aldR, AdhP1, AdhP2 and dkgA；The gene akR of aldehyde ketone reductase is encoded as contained SEQ ID NO.1
Shown in sequence, the gene aldR for encoding alcohol dehydrogenase contains the sequence as shown in SEQ ID NO.2, encodes the base of alcohol dehydrogenase
Because AdhP1 contains the sequence as shown in SEQ ID NO.3, the Gene A dhP2 for encoding alcohol dehydrogenase contains such as SEQ ID NO.4 institutes
The gene dkgA of the sequence shown, coding 2,5-, bis- dehydrogenation gluconic acid reductases contains the sequence as shown in SEQ ID NO.5.
3. recombination according to claim 1 or 2 solves ornithine Raoul bacterium, which is characterized in that knockout is with Red recombination systems
System is knocked out.
4. a kind of method of recombination solution ornithine Raoul bacterium described in structure claim 1, which is characterized in that specific steps are such as
Under：
(1) it using pKD13 plasmids as template, separately designs same containing the both sides target gene akR, aldR, AdhP1, AdhP2 or dkgA
The primer amplification of source segment goes out to knock out the target practice segment of target gene；
(2) any target practice segment electrotransformation obtained in (1) is entered into the pKD46 plasmids containing chloramphenicol resistance gene
In R.ornithinolytica BF60, it is coated with the tablet containing kalamycin resistance, positive gram is obtained by bacterium colony PCR verifications
Grand bacterial strain；
(3) positive colony bacterium obtained in high-temperature cultivation (2), loses temperature sensitive type pKD46 plasmids, and then electrotransformation enters
PCP20 plasmids, high-temperature cultivation eliminate kalamycin resistance gene label, are filtered out while being lost using different resistant panels
The mutant strain of pCP20 plasmids and kalamycin resistance gene label, this mutant strain is what target gene lacked
R.ornithinolytica；
(4) step (1)~(3) are repeated, to knock out one or more of five 5 hydroxymethyl furfural reductase genes.
5. a kind of method improving solution ornithine Raoul bacterium 2,5-furandicarboxylic acid yield, which is characterized in that solve ornithine
Raoul bacterium (Raoultella ornithinolytica) BF60 is starting strain, knocks out five 5 hydroxymethyl furfural reductases
In at least three；Five 5 hydroxymethyl furfural reductases are by following gene code：akR、aldR、AdhP1、AdhP2、
dkgA。
6. a kind of method producing 2,5-furandicarboxylic acid, which is characterized in that recombinate solution bird ammonia so that claims 1 to 3 is any
The resting cell of sour Raoul bacterium is biocatalyst, using 5 hydroxymethyl furfural as substrate, catalytic production 2,5-furandicarboxylic acid.
7. according to the method described in claim 6, it is characterized in that, according to 1.5~5g cells/g 5 hydroxymethyl furfurals dosage
Carry out catalysis reaction.
8. the method described according to claim 6 or 7, which is characterized in that it is described catalysis be at 20~37 DEG C reaction 12~
60h。
9. according to any method of claim 6~8, which is characterized in that the recombination solution ornithine Raoul bacterium is pressed
State step culture acquisition：
(1) seed liquor culture：Picking recombination solution ornithine Raoul bacterium single bacterium colony is inoculated in LB liquid medium, and 28~30
DEG C, culture 10~12h；
(2) thalline culture：Seed liquor is transferred to 1~2% inoculum concentration in TB culture mediums, 28~30 DEG C of 16~36h of culture.
10. any recombination solution ornithine Raoul bacterium of claims 1 to 3 prepares in food, biology, chemical field contains 2,5-
Application in terms of the product of furandicarboxylic acid.
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