CN105002204B - One plant height produces the Gluconobater oxydans genetic engineering strain and its preparation method and application of 5-KGA - Google Patents
One plant height produces the Gluconobater oxydans genetic engineering strain and its preparation method and application of 5-KGA Download PDFInfo
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Abstract
The invention discloses a plant height produce 5 KGA Gluconobater oxydans genetic engineering strain and its preparation method and application,（1）The upstream and downstream segment of GA2DH genes and PDC genes is introduced into recombination and integration type suicide plasmid respectively, derives from kill knockout plasmid p △ GOX1231 and p △ GOX1081 respectively；（2）P △ GOX1231 are transferred in host strain, are screened successively through antibiotic resistance screening, nonreactive culture and sucrose, Positive mutants of GA2DH gene knockouts is then obtained by bacterium colony PCRG.oxydansZJU1；P △ GOX1081 are transferred to by same methodG.oxydansPositive mutants of PDC gene knockouts is obtained in ZJU1G.oxydansZJU2；（3）It willsldAB、pqqABCDE、tldD、P0169And cytochromesbo 3Oxidizing fermentcyoBACD genes are transferred to step（2）Gained Gluconobater oxydans genetic engineering strainG.oxydansIn ZJU2 to obtain the final product.GA2DH is the Major Enzymes that Gluconobacter oxydans form 2 KGA, and PDC forms that acetaldehyde is related then with catalysis pyruvate decarboxylation, and knockouts of GA2DH and PDC can solve the shunting of the metabolism in 5 KGA forming processes, reduce by-product, the high yield of 5 KGA of realization.
Description
Technical field
The present invention relates to a kind of gluconobacter suboxydans Gluconobacter genetic engineering bacteriums and its applications, especially table
Up to film combination sorbitol dehydrogenase and coenzyme PQQ and respiratory chain terminal electron acceptor, 5- keto-D-gluconic acids (5- is improved
Keto-D-gluconic acid, 5-KGA) engineering bacteria and application, belong to genetic engineering and fermentation engineering field.
Background technology
5- keto-D-gluconic acids (5-keto-D-gluconic acid, 5-KGA) are a kind of important intermediate compounds
Object.Phase late 1940s, Gray synthesizes Vc by 5-KGA, and applies for a patent US2421611, US2421612.Later again
There is patent report 5-KGA to synthesize xylose diacid, such as US5731467 synthesizes aromatic substance 4- hydroxy-5-methyl base -2,3- dihydroxy
Furanone, such as US4464409.More important is 5-KGA to prepare L- (+)-tartaric acid (Journal of by chemical catalysis
American Chemical Society, 1933,55,3563, US2380196, WO9615095A1, GB2388368).Oxidation
Gluconobacter suboxydans (Gluconobacter oxydans) 621H is resistant to the glucose of high concentration, and using glucose as substrate,
Incomplete oxidation is gluconic acid (gluconic acid, GA), is further oxidized to 2-KDG (2-keto-
D-gluconic acid, 2-KGA) or 5-KGA.
The German research centers J ü lich carry out genetic modification, such as GA2DH to Gluconobacter oxydans DSM2343
The inactivation of gene (gluconic acid -2- dehydrogenases (gluconate-2-dehydrogenase) gene), the table excessively of GA5DH genes
Reach, express the transformation etc. of promoter, structure engineering bacteria G.oxydans MF1/pBBR1MCS5-PtufB- ga5dh, final 5-KGA
Yield reach 300mM, about 58.2g/L (Applied Microbiology and Biotechnology, 2006,73 (2),
443-451).Japanese mountain pass university Saichana etc. screens one plant of heat resistance Gluconobacter, and is inserted into resistant gene mistake
GA2DH living, final 5-KGA yield are 190mM, about 36.86g/L (Appiled and Enviromental
Microbiology,2009,75(13),4240-4247).Zhejiang University Li Boyi etc. studies G.oxydans CGMCC 1.637
Technological condition for fermentation under conditions of dissolved oxygen control 15%, stream plus mend glucose, final 5-KGA yield is reached in constant pH 5.5
To 75.5g/L, conversion ratio is more than 70% (bioengineering journal, 2014,30 (9), 1486-1490)；University Of Science and Technology Of Tianjin Tan it
Of heap of stone to wait using the Gluconobacter oxydans HGI-1 of 2-KGA deficiencies as starting strain, research carbon and nitrogen sources close 5-KGA
At influence, batch fermentation scale-up is carried out in 5L fermentation tanks, the yield of 5-KGA is 93.80g/L, and average generating rate is
1.56g/L·h。
The genome sequence of Gluconobacter oxydans 621H in 2005 announces (NC_006677), Nature
Biotechnology,23(2),195-200.It participates in glucose metabolism and generates 2 key enzymes of 5-KGA to be membrane-bound protein respectively
White grape glucocorticoid dehydrogenase (membrane-bound glucose dehydrogenase, mGDH) and film combination sorbitol dehydrogenase
(membrane-bound sorbitol dehydrogenase, SLDH), with pyrroloquinoline quinone
(pyrroloquinoline quinone, PQQ) is coenzyme, and transmission electronics most passes through afterwards to respiratory chain ubiquinone (ubiquinone)
Terminal electron acceptor cytochrome b o3Oxidizing ferment (cytochrome bo3Oxidase it) aoxidizes, electronics is made to be passed to point from ubiquinone
Sub- oxygen, and generate proton gradient potential.2013, Meyer and Richhardt had studied coenzyme PQQ and cytochrome b o respectively3
Effect of the oxidizing ferment when being overexpressed embrane-associated protein finds an important factor for amount of PQQ is influence dehydrogenase oxidoreductase substrate, and
Cytochrome b o3Oxidizing ferment is rate-limiting factor (the Applied Microbiology and of respiratory chain electron transmission
Biotechnology,2013,97,3457-3466；Journal of Bacteriology,2013,195(18),4210-
4220).2013, Chen Jian etc. applied for a patent CN 103484417A, the expressing K .vulgare in G.oxydans WSH-003
SDH, SNDH and the PQQ in the sources WSH-001, one-step method is from sorb alcohol production 2-keto-L-gulonic acid (2-keto-L-gulonic
acid,2-KLG).The same year, Tianjin member English express sorbose dehydrogenation simultaneously into equal in Ketogulonigenium vulgare
Enzyme, sorb ketose dehydrogenase and coenzyme PQQ, from sorbose production 2-KLG (Metabolic Engineering
2013,19,50-56)。
Invention content
The present invention is directed to the problem of existing 5-KGA low outputs, provides the Gluconobacter oxvdans of plant height production 5-KGA
Genetic engineering bacterium and its preparation method and application,
One plant height produces the preparation method of the Gluconobater oxydans genetic engineering strain of 5-KGA, includes the following steps：
(1) by GA2DH genes and PDC genes (pyruvate decarboxylase (pyruvate decarboxylase) gene)
Upstream and downstream segment introduces the multiple cloning sites of recombination and integration type suicide plasmid pJKM respectively, respectively obtains the suicide of GA2DH genes
The suicide type that type knocks out plasmid p Δ GOX1231 and PDC genes knocks out plasmid p Δs GOX1081；The recombination and integration type suicide matter
Grain carries SacB genes, antibiotic marker and multiple cloning sites；
(2) gained suicide type knockout plasmid p Δ GOX1231 electrotransformations are transferred in host strain, successively through antibiotic resistance
Then screening, nonreactive culture and sucrose screening are obtained Positive mutants of GA2DH gene knockouts by bacterium colony PCR, are named as
G.oxydans ZJU1；
(3) gained suicide type knockout plasmid p Δ GOX1081 electrotransformations are transferred to the G.oxydans ZJU1 after passage
In, it is screened successively through antibiotic resistance screening, nonreactive culture and sucrose, the sun of PDC gene knockouts is then obtained by bacterium colony PCR
Property muton is named as G.oxydans ZJU2 to get Gluconobater oxydans genetic engineering strain；
(4) by sldAB, pqqABCDE, tldD, P0169And cytochrome b o3Oxidizing ferment cyoBACD genes are transferred to step (3)
In gained Gluconobater oxydans genetic engineering strain G.oxydans ZJU2 to obtain the final product.
The present invention is overexpressed film combination sorbitol dehydrogenase sldAB genes and amalgamation and expression coenzyme PQQ bases in host
Because of cluster and terminal electron acceptor cytochrome b o3Oxidizing ferment.Genetic engineering bacterium is prepared based on the seamless system of gene
G.oxydans ZJU2, by controlling pH, dissolved oxygen, under conditions of fed-batch fermentation, 5-KGA yield reaches 102g/L, average to generate
Rate is 1.7g/Lh.However, the yield of 5-KGA biosynthesis is opposite or relatively low, wanting for industrial applications cannot be satisfied
It asks, for example 5-KGA is used for being catalyzed preparation L- (+)-tartaric acid.To further increase the yield of 5-KGA, the present invention exists
On the basis of G.oxydans ZJU2, one plant of overexpression sldAB gene is built by genetic engineering means, and amalgamation and expression is auxiliary simultaneously
Enzyme PQQ and terminal electron acceptor cytochrome b o3The engineering bacteria of oxidizing ferment, energy Efficient Conversion glucose are 5-KGA.Using auxiliary
Transformation produces 5-KGA in Gluconobacter oxvdans and has not been reported at home while enzyme and respiratory chain metabolic engineering.
After passage, suicide plasmid will be eliminated G.oxydans ZJU1 automatically, not influence the modification of next step gene.
The base sequence of the SacB genes is as shown in SEQ ID NO.1.
Preferably, the antibiotic marker is kalamycin resistance gene marker.
The preparation method of preferably recombination and integration type suicide plasmid is as follows：
(1) the cloned plasmids pEASY-Blunt-SacB of the gene containing SacB is built；
(2) the correct cloned plasmids pEASY-Blunt-SacB of sequencing is taken, SspI single endonuclease digestion cloned plasmids pEASY- is passed through
Blunt-SacB and suicide plasmid pK18mobGII is recycled and is connected, is transformed into E.coli DH5 α, and screening obtains SacB connections
Positive colony in the right direction, and E.coli DH5 α are cultivated, extraction integrative plasmid is to get recombination and integration type suicide matter
Grain pJKM.
Suicide plasmid pK18mobGII construction methods of the present invention can be found in document New mobilizable
vector suitable for gene replacement in Gram-negative bacteria and their use
in mapping of the 3’end of the Xanthomonas campestris pv.Campestris gum
operon.Appl Environ Microbiol 1999,65:278-282, the present invention in by the plasmid structure person (IELPI,
L.Instituto de Investigaciones Bioquι′micas Fundacio′n Campomar,Facultad de
Ciencias Exactas y Naturales,UBA,and CONICET,1405 Buenos Aires,Argentina,
Phone:54(1)863-4011/19.Fax:54(1)865-2246.E-mail:LIELPI@iib.uba.ar.) it provides.From certainly
It kills plasmid pK18mobGII to set out, builds the novel integrated suicide plasmid pJKM containing SacB genes, be applied to gluconic acid
The seamless operation of gene of bacillus.One levansucrase that can be secreted of SacB gene codes, energy sucrose hydrolysis form height
The levulan of molecular weight so that lethal phenotype occurs in the sucrose culture medium containing 5-10% in Gram-negative bacteria, is made with this
For reversed selection markers, safe operation is nontoxic.
The method for building the recombinant plasmid pEASY-Blunt-SacB of the gene containing SacB is as follows：
(1) using the base sequence as shown in SEQ ID NO.2 and SEQ ID NO.3 as primer amplification SacB genes；
Sense primer：5’-AATATTcacatatacctgccgttcactat-3’(SEQ ID NO.2)
Downstream primer：5’-AATATTccatcggcattttcttttgcg-3’(SEQ ID NO.3)
(2) by flat end Cloning Kit (pEASY-Blunt Simple Cloning Kit, Quan Shijin, Beijing) into
The flat seamless clone in end of row, obtains the recombinant plasmid pEASY-Blunt-SacB containing SacB genes.
It is wild type Gluconobacter oxvdans that the present invention, which builds host strain described in the step of recombinant bacterium (2),
G.oxydans DSM2343.Host strain is not necessarily to any operation, and wild type is applied widely, safe operation.
Preferably, gluconic acid 2- dehydrogenases (gluconate 2-dehydrogenase, GA2DH) gene and pyruvic acid
The upstream and downstream clip size of dehydrogenase (pyruvate decarboxylase, PDC) gene is 500-1200bp.Further
Preferably 800-1000bp.It is further preferred that GA2DH genes upstream and downstream each 1000bp of genetic fragment；PDC genes are upper and lower
Swim each 800bp of segment.
According to the genome sequence (NC_006677) of the Gluconobacter oxydans 621H of announcement, GA2DH is designed
With the primer of PDC gene upstream and downstream homologous fragments：
The primer of GA2DH gene upstream and downstream homologous fragments：
1231_HindIII_F：5’-ataAAGCTTagccaaaggcggaaagacggc-3’(SEQ ID NO.4)
1231_Fus_R：5’-catttcaggggagaccgcttaaatgaagtggccgctggtcatc-3’(SEQ ID
NO.5)；
1231_Fus_F：5’-gatgaccagcggccacttcatttaagcggtctcccctgaaatg-3’(SEQ ID
NO.6)
1231_XbaI_R：5’-ataTCTAGAcgccggcactttcttctacc-3’(SEQ ID NO.7)；
The primer sequence for expanding the upstream and downstream PDC genetic fragment is as follows：
1081_HindIII_F：5’-cccAAGCTTctcgtctgggcgattcatg-3(SEQ ID NO.8)
1081_Fus_R：5’-Cctgaggtactgaaatcatgacaaagcgtctgatccttcc-3(SEQ ID NO.9)
1081_Fus_F：5’-Ggaaggatcagacgctttgtcatgatttcagtacctcagg-3(SEQ ID
NO.10)
1081_SalI_R：5’-acgcGTCGACaggcatgagacctacctga-3(SEQ ID NO.11).
PCR obtains target fragment and is integrated into plasmid pJKM respectively then by fusion DNA vaccine, builds respective suicide
Knock out plasmid p Δ GOX1231 and p Δs GOX1081.
Preferably, the antibiotic resistance screening is that transformant is coated on kanamycins 50 μ g/m and 50 μ of Cefoxitin
The seed culture medium tablet of g/m obtains genome conformity type transformant；The sucrose screening is antibiotic resistance gene integration transformation
After mannitol culture medium Tube propagation, the seed culture containing 5~10% (preferably 10%) sucrose and Cefoxitin is lined
Base tablet obtains the saltant for being detached from suicide plasmid.
The nonreactive culture is the recon that will obtain through 30 DEG C of overnight incubations of nonreactive.
By sldAB, pqqABCDE, tldD, P in step (4)0169And cytochrome b o3Oxidizing ferment cyoBACD genes are transferred to
Method in Gluconobater oxydans genetic engineering strain G.oxydans ZJU2 obtained by step (3) preferably includes following steps：
(1) PCR obtains sldAB, pqqABCDE, tldD, P0169And cytochrome b o3Oxidizing ferment cyoBACD genetic fragments；
(2) by sldAB genes and P0169Structure recombinant expression plasmid pBB5-P is connect with pBBR1MCS5 carriers0169-
sldAB；By pqqABCDE, tldD, cytochrome b o3Oxidizing ferment cyoBACD and promoter P0169It connect structure with carrier pUCpr
Recombinant expression plasmid pUCpr-P0169-pqqABCDE-tldD-P0169-cyoBACD；
(3) two kinds of recombinant expression plasmids are transferred to by electrotransformation in G.oxydans ZJU2.
It is further preferred that according to Gluconobacter oxydans 621H genome sequences (NC_ in GenBank
006677), design primer：
SLDH_F:’-GCTCTAGAGGACTTTCAGTTCTGGAGGCTTTCACCA-3’(SEQ ID NO.12)
SLDH_R:5’-CGGAATTCTCCCACCCGAAAAATGGAAAAAACG-3’(SEQ ID NO.13)
After PCR obtains SLDH gene sldAB segments, it is connected to pEASY-Blunt cloning vectors and is sequenced, meanwhile, in the future
Derived from the promoter P of Gluconobacter oxydans GOX0169 genes0169It is cloned in pEASY-Blunt cloning vectors simultaneously
Sequencing, progress double digestion is connected to wide host expression vector pBBR1MCS5 after obtaining correct transformant, builds expression plasmid
pBB5-P0169- sldAB, the recombinant expression plasmid Transformed E .coli DH5 α that will be built, and verify positive transformants through bacterium colony PCR
There are 103bp and 2771bp bands respectively in son；
Promoter P0169Amplimer：
0169_SacI_F：5’-ATAGAGCTCtgaaagcggctggcgcgt-3’(SEQ ID NO.14)；
0169_XbaI_R：5’-GCTCTAGAgcggaaggcgttataccctga-3’(SEQ ID NO.15)。
PQQ gene clusters pqqABCDE, tldD, the cell color that will be annotated in G.oxydans DSM2343 genomes
Plain bo3Oxidizing ferment cyoBACD and promoter P0169, separately design primer and obtain gene, wherein each segment contains and is correspondingly connected with
The overlapping regions 15-20bp are connected respectively to pEASY-Blunt cloning vectors and are sequenced, after obtaining correct segment, using full formula
Gold orients seamless Cloning Kit (number：CU101 seamless clone, structure recombinant expression plasmid pUCpr-P) are carried out0169-
pqqABCDE-tldD-P0169-cyoBACD；
Promoter, fusion P0169Amplimer：
Add_0169_F：5’-acactgtttaaacaccgtgaaagcggctggcgc-3’(SEQ ID NO.16)
pQQ_Fuse0169_R：5’-acatccgcgcggaaggcgttatac-3’(SEQ ID NO.17)；
Merge the amplimer of pqqABCDE：
pQQ_Fuse0169_F：5’-ccttccgcgcggatgttcagg-3’(SEQ ID NO.18)
tldD_FusepQQ_R：5’-ccggctagaagatggcctctc-3’(SEQ ID NO.19)；
Merge the amplimer of tldD：
tldD_FusepQQ_F：5’-gccatcttctagccggtctgttc-3’(SEQ ID NO.20)
0169_FusetldD_R：5’-ctttcaggatcttcttcatg-3’(SEQ ID NO.21)；
Merge Article 2 promoter P0169Amplimer：
0169_FusetldD_F：5’-tcgcgactgaaagcggctggc-3’(SEQ ID NO.22)
ADD_0169_R：5’-cggtacccggggatcctgcggaaggcgttatac-3’(SEQ ID NO.23)；
The amplimer of cyoBACD：
cyoBACD_XbaI_F：5’-cgatTCTAGAactactgcaagccggaacgg-3’(SEQ ID NO.24)
cyoBACD_SacI_R：5’-actgGAGCTCaagggctggcaggatttctc-3’(SEQ ID NO.25).
The two recombinant expression plasmid pBB5-P built0169- sldAB and pUCpr-P0169-pqqABCDE-tldD-
P0169- cyoBACD is transferred to G.oxydans ZJU2, structure recombination engineering G.oxydans by the method for electrotransformation twice
ZJU2/pBB5-P0169-sldAB/pUCpr-P0169-pqqABCDE-tldD-P0169-cyoBACD。
The present invention also provides a kind of Gluconobacter oxvdans bases for the high yield 5-KGA being prepared by the preparation method
Because of engineering bacteria.The genetic engineering bacterium of the present invention can express sldAB, pqqABCDE-tldD and cyoBACD in the cell, and
5-KGA is produced for transforming glucose.Recombinant expression plasmid constructed by the present invention is composing type, during thalli growth
It can synchronous expression.
The present invention also provides a kind of if the Gluconobater oxydans genetic engineering strain of the high yield 5-KGA is in production 5-
Application in KGA.
Preferably, the application includes the following steps：
By the high yield 5-KGA Gluconobater oxydans genetic engineering strains through expand cultivate after be seeded in fermentation tank into
Row fermented and cultured collects zymotic fluid.Detect the 5- keto-D-gluconic acid yield in zymotic fluid.
5- keto-D-gluconic acids in zymotic fluid are used to prepare L-TARTARIC ACID after can further isolating and purifying.
The group of the fermentation medium becomes：Corn steep liquor 7.5g/L；(NH4)2SO40.41g/L；(NH4)2HPO4 0.1g/
L；MgSO4·7H2O 0.07g/L；CaCO310-20g/L (is separately added after sterilizing)；Glucose 100g/L, initial pH 5.0-
5.5,121 DEG C, sterilize 15min, 50 μ g/mL of Cefoxitin final concentration.
The condition of fermented and cultured is：The condition of fermented and cultured is：28~32 DEG C, the ventilatory capacity of 0.8~1.2vvm, tank pressure
0.02~0.04MPa, mixing control oxyty are 20%~40%, and ferment 55~65h, according in culture medium in fermentation process
Concentration of glucose control feed supplement.
It is further preferred that the application includes the following steps：
5mL seed culture medium test tubes are added in the 200 μ L of recombinant bacterium of glycerol tube preservation, and in 30 DEG C, 200rpm overnight incubations are pressed
4% (V/V) inoculum concentration transfers 50mL fresh seeds culture medium to exponential phase (about 10-14h), turns by 8% (V/V) inoculum concentration
200mL fresh seeds culture medium is connect to exponential phase；It is inoculated in 15L fermentation tank (working volumes by 8% (V/V) inoculum concentration
9L), 30 DEG C, the ventilatory capacity of 1vvm, tank presses 0.03MPa, speed of agitator to control dissolved oxygen, and ferment 60h.
Seed culture medium (g/L)：Mannitol 25；Yeast extract 5；tryptone 3；15 (solid culture of agar
Base)；pH 5.5-6.0；121 DEG C of sterilizings 20min, the western 50 μ g/mL of spit of fland final concentration of cephalo.
Glucose need to carry out feed supplement in fermentation process, and feed supplement condition is：When in culture medium concentration of glucose be less than 10g/L,
It is more excellent to start to fill into glucose when concentration of glucose is most preferably less than 30g/L less than 20g/L with concentration of glucose, and
It controls remaining sugar concentration in fermentation tank and is not less than 30g/L, the amount of filling into of glucose is not higher than the 40% of initial glucose amount.
Most preferably, the condition of fermented and cultured is：30 DEG C, the ventilatory capacity of 1vvm, tank presses 0.03MPa, mixing control dissolved oxygen
A concentration of 20%~40%, according to dissolved oxygen situation, mixing speed control is in 200rpm~800rpm, and ferment 60h, in fermentation process
Feed supplement is controlled according to the concentration of glucose in culture medium, concentration of glucose starts to fill into glucose when being less than 30g/L, and
It controls remaining sugar concentration in fermentation tank and is not less than 30g/L, the amount of filling into of glucose is not higher than the 40% of initial glucose amount.
Glucose, GA and 5-KGA assays in the present invention：Fermentation broth sample crosses 0.22 μm after 12000rpm is centrifuged
Filter membrane processing.GA and 5-KGA is measured using high performance liquid chromatography (HPLC).1100 chromatographs of Agilent, Showa electrician
Shodex DE-613 chromatographic columns, mobile phase：2mM perchloric acid solutions, flow velocity：0.5mL/min, sample size：10 μ L, 210nm are ultraviolet
Detection.Glucose uses bio-sensing analyzer SBA-40D.
The present invention is by the seamless modification of gene, under the premise of without any exogenous dna fragment, knocks out
Gluconic acid 2- dehydrogenases in Gluconobacter oxydans DSM2343 (gluconate2-dehydrogenase,
GA2DH) and pyruvic dehydrogenase (pyruvate decarboxylase, PDC) gene, a plant weight group genetic engineering bacterium is built
G.oxydans ZJU2 solve the problems, such as GM microbial safeties, and it can transforming glucose be effectively 5-KGA；For into
One step improves the yield of 5-KGA, and the present invention builds one plant on the basis of G.oxydans ZJU2, by genetic engineering means and crosses table
Up to sldAB genes, and amalgamation and expression coenzyme PQQ and terminal electron acceptor cytochrome b o simultaneously3The engineering bacteria of oxidizing ferment, energy
Efficient Conversion glucose is 5-KGA.
The present invention has the beneficial effect that：
GA2DH is that Gluconobacter oxydans form 2-KDG (2-keto-D-gluconic acid, 2-
KGA Major Enzymes), and PDC forms acetaldehyde correlation then with catalysis pyruvate decarboxylation, therefore, the knockout of GA2DH and PDC can solve
The certainly metabolism shunting in 5-KGA forming processes, reduces by-product, realizes the high yield of 5-KGA.
The present invention has been knocked out GA2DH the and PDC genes of G.oxydans DSM2343, has been weighed by the seamless modification of gene
Group bacterium G.oxydans ZJU2.Further by the transformation of genetic engineering, in the genetic engineering bacterium G.oxydans ZJU2 of preparation
Middle expression sldAB, pqqABCDE-tldD and cyoBACD gene, structure recombinant expression plasmid pBB5-P0169- sldAB and
pUCpr-P0169-pqqABCDE-tldD-P0169- cyoBACD, main application are to improve the yield of 5-KGA.It ferments in 15L
On tank, dissolved oxygen technique is controlled using fed batch fermentation, 5-KGA yield reaches 162g/L, and yield 2.53g/Lh has had work
The value of industry application.
Description of the drawings
Fig. 1 is the novel integrative plasmid pJKM of structure.
Fig. 2 is principle and the flow signal of GA2DH gene knockouts.
Fig. 3 is the bacterium colony PCR the selection result figures of recombinant bacterium G.oxydans ZJU1.
Fig. 4 is the bacterium colony PCR the selection result figures of recombinant bacterium G.oxydans ZJU2.
Fig. 5 is genetic engineering bacterium G.oxydans ZJU2/pBB5-P0169-sldAB/pUCpr-P0169-pqqABCDE-
tldD-P0169- cyoBACD course of fermentation curve graphs.
Specific implementation mode
Seed culture medium (g/L)：Mannitol 25；Yeast extract 5；tryptone 3；15 (solid culture of agar
Base)；pH 5.5-6.0；121 DEG C of sterilizings 20min, the western 50 μ g/mL of spit of fland final concentration of cephalo.
Fermentation medium (g/L)：Corn steep liquor 7.5；(NH4)2SO40.41；(NH4)2HPO40.1；MgSO4·7H2O
0.07；CaCO310-20 (is separately added after sterilizing)；Glucose 25-100 (as needed), initial pH 5.0-5.5,121 DEG C,
Sterilize 15min, the western 50 μ g/mL of spit of fland final concentration of cephalo.
The experimental methods of molecular biology of actual conditions is not specified in following embodiment, according to normal condition, reference《Point
Sub- cloning experimentation guide》(New York：Cold Spring Harbor Laboratory Press, 2001) condition described in
It carries out.
The structure of 1 novel integrative plasmid pJKM of embodiment
According to plasmid pSM20 design primers (base sequence is as shown in SEQ ID NO.2 and SEQ ID NO.3), PCR amplification
Obtain SacB genetic fragments, by flat end Cloning Kit (pEASY-Blunt Simple Cloning Kit, Quan Shijin,
Beijing) the seamless clone in flat end is carried out, obtain the recombinant plasmid pEASY-Blunt-SacB containing SacB genes.
After sequencing is correct, SspI single endonuclease digestions cut recycling SacB genetic fragments, and with the pK18mobGII through SspI single endonuclease digestions
Plasmid is connected, Transformed E .coli DH5 α, and bacterium colony PCR screenings obtain the correct positive colony in SacB connections direction, are built
Integrated suicide plasmid pJKM, plasmid map is as shown in Figure 1.
2 target gene of embodiment knocks out the structure of suicide plasmid
According to the genome sequence (NC_006677) of Gluconobacter oxydans 621H in GenBank, set respectively
Primer is counted, primer sequence is as shown in SEQ ID NO.4~SEQ ID NO.7：
PCR obtains GA2DH upstream and downstream each 1000bp of genetic fragment, and passes through fusion DNA vaccine (the upper and lower piece obtained with PCR
Section is template, and 1231_HindIII_F and 1231_XbaI_R are primer, carry out the fusion piece that secondary PCR obtains upstream and downstream connection
Section) it is integrated into integrated plasmid pJKM (preparation of embodiment 1), structure suicide knocks out plasmid p Δ GOX1231, sequence verification of going forward side by side
Correctly.
The primer sequence of the upstream and downstream PDC genetic fragment is expanded as shown in SEQ ID NO.8~SEQ ID NO.11, is used
The primer, PCR obtain PDC upstream and downstream each 800bp of genetic fragment, and building suicide using identical method knocks out plasmid p Δs
GOX1081 after sequence verification of going forward side by side is correct, carries out next step operation.
The structure of 3 engineering bacteria G.oxydans ZJU2 of embodiment
The knockout plasmid p Δs GOX1231 built is transferred to recipient bacterium Gluconobacter oxvdans by electrotransformation
In Gluconobacter oxydans DSM2343 (being purchased from Germany Biological Resource Center DSMZ), and it is coated on containing kanamycins
The seed culture medium tablet of 50 μ g/mL of 50 μ g/mL and Cefoxitin carries out single-swap screening, obtains suicide plasmid and be integrated into base
Because of the transformant of group.
The switching of obtained single-swap transformant is stayed overnight in the seed culture medium Tube propagation of nonreactive again, and is lined containing head
The seed culture medium tablet of the western fourth of spore and 10% sucrose carries out the second wheel screening, obtains double crossing over transformant, is converted in double crossing over
In son, the recombinant bacterium G.oxydans ZJU1 of GA2DH gene knockouts are screened by bacterium colony PCR.
.The principle and flow of GA2DH gene knockouts illustrate the bacterium colony PCR as shown in Fig. 2, recombinant bacterium G.oxydans ZJU1
Qualification figure is as shown in figure 3, by the selection result of Fig. 3 it is found that successfully will in recombinant bacterium G.oxydans ZJU1
GA2DH gene knockouts in Gluconobacter oxydans DSM2343.
On this basis, PDC genes are knocked out by identical method, obtains engineering bacteria G.oxydans ZJU2, recombinant bacterium
The bacterium colony PCR qualification figures of G.oxydans ZJU2 as shown in figure 4, the selection result of Fig. 4 it is found that recombinant bacterium G.oxydans ZJU1
In successfully by the PDC gene knockouts in Gluconobacter oxydans DSM2343.
The structure of 4 recombinant expression plasmid of embodiment
(1)pBB5-P0169The structure of-sldAB plasmids
According to genome sequence (NC_006677) design of Gluconobacter oxydans 621H in GenBank
SldAB gene primers, sequence is as shown in SEQ ID NO.12 and SEQ ID NO.13：
PCR obtains SLDH gene sldAB segment 2771bp, and is cloned in pEASY-Blunt carriers.After being sequenced correctly,
EcoRI and XbaI double digestions recycle sldAB segments and are connected to wide host expression vector pBBR1MCS5, structure plasmid pBB5-
sldAB；Design primer (its sequence is as shown in SEQ ID NO.14 and SEQ ID NO.15) PCR acquisitions derive from
The promoter P of Gluconobacter oxydans GOX0169 genes0169Segment, and pEASY-Blunt carriers are cloned in, it passes through
After sequencing is correct, double digestion recycles P0169Segment is simultaneously inserted into pBB5-sldAB, structure expression plasmid pBB5-P0169-sldAB。
The recombinant expression plasmid Transformed E .coli DH5 α that will be built, and verify positive transformant through bacterium colony PCR, that is, divide
Do not occur 103bp and 2771bp bands.
(2)pUCpr-P0169-pqqABCDE-tldD-P0169The structure of-cyoBACD plasmids is according in GenBank
Gluconobacter oxydans 621H genome sequence (NC_006677) design pqqABCDE, tldD, cyoBACD with
And promoter P0169Gene primer, in order to realize that polygenic connection, each genetic fragment that PCR is obtained contain 15-20bp respectively
Overlapping region.Each segment that PCR is obtained through be sequenced it is correct after, using full formula gold orient seamless Cloning Kit (Seamless Cloning and Assembly Kit, CU101) connection structure recombinant expression plasmid
pUCpr-P0169-pqqABCDE-tldD-P0169, with cyoBACD genes by digestion, recycling, connect, build expression plasmid
pUCpr-P0169-pqqABCDE-tldD-P0169-cyoBACD(P0169、pqqABCDE、tldD、P0169With the primer sequence of cyoBACD
Row are as shown in SEQ ID NO.16~SEQ ID NO.25), wherein operation is carried out by kit specification requirement.
Embodiment 5
G.oxydans ZJU2/pBB5-P0169-sldAB/pUCpr-P0169-pqqABCDE-tldD-P0169- cyoBACD works
The structure of journey bacterium
By the Gluconobacter oxydans ZJU2 built in embodiment 3 in electrotransformation culture medium (mannitol
80g/L, yeast extract 15g/L, MgSO4·7H2O 2.5g/L, glycerine 0.5g/L, CaCl2It is cultivated extremely in 1.5g/L)
OD600For 0.4-0.6,4 DEG C, thalline were collected by centrifugation by 6000 × g, 4min, and the sterile glycerol solution of 10% (V/V) is iced with 30mL
It is resuspended, after being repeated 2 times, thalline is resuspended in the sterile glycerol solution that 2mL ices 10% (V/V), and 40 μ L, mono- pipes are packed as experiencing
State cell.pBB5-P0169- sldAB plasmids by electrotransformation into G.oxydans ZJU2 competent cells, and containing 50 μ g/mL
Positive recombinant G.oxydans ZJU2/pBB5-P are screened on the mannitol tablet of gentamicin0169-sldAB。
Correct positive recombinant G.oxydans ZJU2/pBB5-P will be obtained0169- sldAB, and make according to the method described above
Standby competent cell.Again by electrotransformation method, by pUCpr-P0169-pqqABCDE-tldD-P0169- cyoBACD is transferred to
G.oxydans ZJU2/pBB5-P0169- sldAB, and containing 50 μ g/mL gentamicins and 100 μ g/mL ampicillins
Positive recombinant is screened on mannitol tablet, obtains recombination engineering G.oxydans ZJU2/pBB5-P0169-sldAB/
pUCpr-P0169-pqqABCDE-tldD-P0169-cyoBACD。
Embodiment 6
G.oxydans ZJU2/pBB5-P0169-sldAB/pUCpr-P0169-pqqABCDE-tldD-P0169- cyoBACD is sent out
Ferment produces 5-KGA
Seed culture medium (g/L)：Mannitol 25；Yeast extract 5；tryptone 3；pH5.5-6.0；121 DEG C go out
100 μ g/mL of bacterium 20min, 50 μ g/mL of gentamicin final concentration and ampicillin final concentration.
Fermentation medium (g/L)：Corn steep liquor 7.5；(NH4)2SO40.41；(NH4)2HPO40.1；MgSO4·7H2O
0.07；CaCO310-20 (is separately added after sterilizing)；Initial glucose 100, initial pH 5.0-5.5,121 DEG C, sterilizing
100 μ g/mL of 15min, 50 μ g/mL of gentamicin final concentration and ampicillin final concentration.
Condition of culture：5mL seed culture medium test tubes are added in the 200 μ L of recombinant bacterium of glycerol tube preservation, and in 30 DEG C, 200rpm is trained
It supports overnight, 50mL fresh seeds culture medium is transferred to exponential phase (about 10-14h), by 8% (V/ by 4% (V/V) inoculum concentration
V) inoculum concentration transfers 200mL fresh seeds culture medium to exponential phase；It is inoculated in 15L fermentation tanks by 8% (V/V) inoculum concentration
(working volume 9L), 30 DEG C, the ventilatory capacity of 1vvm, tank presses 0.03MPa, speed of agitator to control dissolved oxygen 20-60%.When in culture medium
Concentration of glucose starts to fill into glucose when being less than 30g/L, and controls remaining sugar concentration in fermentation tank and be not less than 30g/L.
The amount of filling into of glucose is 700g.Through 64h fermentation ends, the yield of 5-KGA reaches 162g/L, yield 2.53g/Lh, fermentation
Curve is as shown in Figure 5.
Claims (8)
1. a plant height produces the preparation method of the Gluconobater oxydans genetic engineering strain of 5-KGA, which is characterized in that including as follows
Step：
（1）The upstream and downstream segment of GA2DH genes and PDC genes is introduced more grams of recombination and integration type suicide plasmid pJKM respectively
Grand site, the suicide type for respectively obtaining GA2DH genes knock out the suicide type knockout plasmid p of plasmid p △ GOX1231 and PDC genes
△GOX1081；The recombination and integration type suicide plasmid carriesSacBGene, antibiotic marker and multiple cloning sites；The recombination
The preparation method of integrated suicide plasmid pJKM is as follows：
（a）Structure containsSacBThe cloned plasmids p of geneEASY-Blunt-SacB；
（b）Take the correct cloned plasmids p of sequencingEASY-Blunt-SacB, pass throughSspISingle endonuclease digestion cloned plasmids pEASY-
Blunt-SacBWith suicide plasmid pK18mobGII, recycles and connect, is transformed intoE. coliDH5 α, screening obtainSacBConnection
Positive colony in the right direction, and cultivateE. coliDH5 α, extraction integrative plasmid is to get recombination and integration type suicide matter
Grain pJKM；
（2）Gained suicide type knockout plasmid p △ GOX1231 are transferred in host strain, are trained successively through antibiotic resistance screening, nonreactive
It supports and sucrose screening is named as then by Positive mutants of bacterium colony PCR acquisition GA2DH gene knockoutsG. oxydans
ZJU1；Step（2）Described in host strain be wild type Gluconobacter oxvdansG. oxydans DSM2343；
（3）Gained suicide type knockout plasmid p △ GOX1081 are transferred to after passageG. oxydansIn ZJU1, successively through anti-
Then raw element resistance screening, nonreactive culture and sucrose screening obtain Positive mutants of PDC gene knockouts, i.e., by bacterium colony PCR
Gluconobater oxydans genetic engineering strain is obtained, is named asG. oxydansZJU2；
（4）It willsldAB、pqqABCDE、tldD、P0169And cytochromesbo 3Oxidizing fermentcyoBACD genes are transferred to step（3）Gained
Gluconobater oxydans genetic engineering strainG. oxydansIn ZJU2 to obtain the final product.
2. the preparation method of the Gluconobater oxydans genetic engineering strain of high yield 5-KGA according to claim 1, feature
It is, the upstream and downstream clip size of GA2DH genes and PDC genes is 500-1200 bp.
3. the preparation method of the Gluconobater oxydans genetic engineering strain of high yield 5-KGA according to claim 1, feature
It is, the antibiotic resistance screening is the mannitol culture medium flat plate that transformant is coated on to kanamycins and Cefoxitin,
Obtain genome conformity type transformant；The sucrose screening is that antibiotic resistance gene integration transformation is trained through mannitol culture medium test tube
After supporting, the mannitol culture medium flat plate containing 5 ~ 10% sucrose and Cefoxitin is lined, obtains the saltant type for being detached from suicide plasmid
Bacterial strain.
4. the preparation method of the Gluconobater oxydans genetic engineering strain of high yield 5-KGA according to claim 1, feature
It is, step（4）Include the following steps：
（1）PCR is obtainedsldAB、pqqABCDE、tldD、P0169And cytochromesbo 3Oxidizing fermentcyoBACD genetic fragments；
（2）It willsldAB genes and P0169Structure recombinant expression plasmid pBB5-P is connect with pBBR1MCS5 carriers0169-sldAB；It willpqqABCDE、tldD, cytochromesbo 3Oxidizing fermentcyoBACD and promoter P0169Structure recombinant expression is connect with carrier pUCpr
Plasmid pUCpr-P0169-pqqABCDE-tldD-P0169-cyoBACD；
（3）Two kinds of recombinant expression plasmids are transferred to by electrotransformationG. oxydansIn ZJU2.
5. a kind of oxidation grape for the high yield 5-KGA that the preparation method described in claim 1 ~ 4 any claim is prepared
Saccharic acid oxydans genetic engineering bacterium.
6. the Gluconobater oxydans genetic engineering strain of high yield 5-KGA as claimed in claim 5 is in production 5- ketone group-D- grapes
Application in saccharic acid, which is characterized in that include the following steps：
The Gluconobater oxydans genetic engineering strain of the high yield 5-KGA is seeded to after expanding and cultivating in fermentation tank and is carried out
Fermented and cultured collects zymotic fluid, the isolated 5- keto-D-gluconic acids from zymotic fluid.
7. applying according to claim 6, which is characterized in that the group of the fermentation medium of the fermented and cultured becomes：Corn
Starch 7.5 g/L；(NH4)2SO40.41 g/L；(NH4)2HPO40.1 g/L；MgSO4·7H2O 0.07 g/L；CaCO3 10-
20 g/L；Glucose 100 g/L, initial pH 5.0-5.5,121 DEG C, sterilize 15 min, the western 50 μ g/ of spit of fland final concentration of cephalo
mL。
8. applying according to claim 6, which is characterized in that the condition of the fermented and cultured is：28 ~ 32 DEG C, 0.8 ~ 1.2
The ventilatory capacity of vvm, it is 20% ~ 40% that tank, which presses 0.02 ~ 0.04 MPa, mixing control oxyty, and ferment 55 ~ 65h, in fermentation process
Feed supplement is controlled according to the concentration of glucose in culture medium.
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