CN102146415A - Gene knockout bacterium of gluconobacter oxydans and preparation method thereof - Google Patents
Gene knockout bacterium of gluconobacter oxydans and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a gene knockout bacterium of gluconobacter oxydans, a preparation method and application thereof and a gene knockout vector thereby. The preparation method comprises the following steps of: (1) converting escherichia coli by using a gene knockout recombinant vector to obtain a transformant, wherein the gene knockout recombinant vector comprises a gluconobacter oxydans mgdh (Membrane-bound Glutamate Dehydrogenase) gene homologous recombinant front arm, a marker gene and an mgdh gene homologous recombinant back arm in sequence; and (2) carrying out double-parent joint by using the transformant and the gluconobacter oxydans and leading the recombinant vector to enter the gluconobacter oxydans and generate homologous double-exchange with the mgdh gene on a chromosome, wherein an obtained zygote is the mgdh gene knockout bacterium. The mgdh gene knockout bacterium disclosed by the invention has favorable growth by utilizing glucose as a unique carbon source, overcomes the defect that the traditional gluconobacter oxydans only utilizes expensive sorbierite as an effective carbon source, greatly saves the production cost and has significance in the fields of biosynthesis and chiral synthesis.
Description
Technical field
The invention belongs to bioengineering field, the purposes of the clpp gene degerming of clpp gene degerming of particularly a kind of oxidizing glucose acidfast bacilli and preparation method thereof, the recombinant vectors that is used for this gene knockout and this oxidizing glucose acidfast bacilli.
Background technology
Oxidizing glucose acidfast bacilli (Gluconobacter oxydans) is one of biological catalyst of widespread use in the synthetic field of biological industry and chirality.Its maximum characteristics are exactly the ability with a series of glycitols compound of incomplete oxidation, the ketone that reaction generates, aldehyde, acid etc. can direct secretion outside born of the same parents, most reactions all are to be participated in catalyticly by the desaturase on the film, oxidation efficiency is very high.This characteristic makes the oxidizing glucose acidfast bacilli become the desirable strain that microbiological industry is produced.At present, the oxidizing glucose acidfast bacilli has been successfully applied to the industrial production of vitamins C, otan, miglitol, gluconic acid and ketone group gluconic acid etc.
Yet, in practical application and research, find that the suitableeest carbon source of oxidizing glucose acidfast bacilli in growth is sorbyl alcohol.And when being carbon source, often need to add CaCO in the substratum with glucose
3And during the fermentation because film is that gluconic acid makes fermented liquid pH value very low in conjunction with Hexose phosphate dehydrogenase with glucose oxidase, thereby suppressed the growth of cell, need constantly to regulate fermented liquid pH value, these factors make the fermentation of thalline and sepn process all more loaded down with trivial details.Simultaneously in the regulate process of pH, production cost and the microbiological contamination probability is corresponding all raises.But, well-known, the price of sorbyl alcohol than the glucose costliness many, the same about 300 yuan/kg of other sorbyl alcohol market price of AR level, glucose only need 20 yuan/kg, industrial sorbyl alcohol about 4.5 yuan/kg that offers, and cerelose about 2.3 yuan/kg that offers.Yet more up to 80g/L, strengthened the cost of oxidizing glucose acidfast bacilli in industrial application is produced at the used up sorbyl alcohol of the necessary for growth of oxidizing glucose acidfast bacilli so undoubtedly, further enlarged the possibility of producing thereby limited it.Therefore, how reducing the feeding cost of oxidizing glucose acidfast bacilli, is current urgency problem to be solved.At present, mainly concentrating on sorbyl alcohol to the high-density culture of oxidizing glucose acidfast bacilli is on the Optimum of culture medium of main carbon source, makes it utilize glucose fully and reduces that thalline ferments and isolating unnecessary trouble and then the research method that reduces the feeding cost of oxidizing glucose acidfast bacilli still do not have report in glucose and transform the oxidizing glucose acidfast bacilli by genetically engineered.
Summary of the invention
Therefore, the technical problem to be solved in the present invention be exactly the suitableeest carbon source at existing oxidizing glucose acidfast bacilli be expensive sorbyl alcohol, and exist thalli growth bad when being carbon source with cheap glucose, the defective that technology is loaded down with trivial details, a kind of clpp gene degerming of oxidizing glucose acidfast bacilli is provided, it can be sole carbon source with glucose, well-grown.The present invention also provides the purposes of the clpp gene degerming of the preparation method of the clpp gene degerming of this oxidizing glucose acidfast bacilli, the recombinant vectors that is used for this gene knockout and this oxidizing glucose acidfast bacilli.
The present invention solves the problems of the technologies described above one of technical scheme of being adopted: a kind of recombinant vectors that is used for gene knockout, it contains oxidizing glucose acidfast bacilli (Gluconobacter oxydans) film successively in conjunction with Hexose phosphate dehydrogenase (mgdh) dna homolog reorganization forearm, marker gene and mgdh dna homolog reorganization postbrachium.
Among the present invention, described marker gene can be conventional various antibiotic resistance genes, the preferred antibiotics resistant gene, and more preferably gentamicin resistant gene (Gm), more preferably to classify the Genbank accession number as be the 3575th～4608 of U25061.1 to nucleotides sequence.
Among the present invention, the length of described mgdh dna homolog reorganization forearm and postbrachium is 0.5～1.5kb, is respectively that the Genbank accession number is the 180th～1151, the 1186th～2231 of AAW60048 preferably.
Among the present invention, the preferred suicide vector of described recombinant vectors, more preferably plasmid pSUP202.
The present invention solves the problems of the technologies described above two of the technical scheme that adopted: a kind of clpp gene degerming of oxidizing glucose acidfast bacilli, it is the degerming of mgdh clpp gene.
Among the present invention, the Genbank accession number of described mgdh gene is AAW60048.The preferable Genbank accession number that knocked out is the 180th～2231 of AAW60048.
Among the present invention, the preferred oxidation gluconobacter suboxydans of described oxidizing glucose acidfast bacilli 621H.
The present invention solves the problems of the technologies described above three of the technical scheme that adopted: the preparation method of a kind of oxidizing glucose acidfast bacilli mgdh clpp gene degerming may further comprise the steps:
1) with the described recombinant vectors transformed into escherichia coli that is used for gene knockout, obtains transformant;
2) with the transformant of step 1) gained as the donor bacterium, oxidizing glucose acidfast bacilli action receptor bacterium, carrying out two parents engages, described recombinant vectors enters neutralize the mgdh gene generation homology double exchange on its karyomit(e) of oxidizing glucose acidfast bacilli, and the zygote of acquisition is the degerming of mgdh clpp gene.
Among the present invention, described oxidizing glucose acidfast bacilli and colibacillary joint are ordinary methods.Wherein, the transformant of step 1) gained is the donor bacterium, and the oxidizing glucose acidfast bacilli is a recipient bacterium.Intestinal bacteria and oxidizing glucose acidfast bacilli engage as two parents, thereby the recombinant vectors that contains in the transformant is entered in the oxidizing glucose acidfast bacilli.Because described recombinant vectors has the homologous fragment (gdh1 of film in conjunction with Hexose phosphate dehydrogenase, gdh2), therefore can with the mgdhDNA fragment generation homology double exchange on the oxidizing glucose acidfast bacilli karyomit(e), the mgdh fragment that will contain the Gm resistant gene is incorporated on the karyomit(e) of oxidizing glucose acidfast bacilli, thereby make film in conjunction with the glucose dehydrogenase gene inactivation, the zygote of gained is the degerming of mgdh clpp gene.And the host e. coli of described transformant can be the bacterial strain in colibacillary K-12 strain source, DH5 α for example, JM109, preferred E.coli JM109.
Preferable, as the routine techniques of this area, step 2) can also add in and help bacterium, carry out three parents and engage.Described help bacterium is to contain the intestinal bacteria that help plasmid pRK2013 or PRK2073.This contains the bacterial strain that the intestinal bacteria that help plasmid can be colibacillary K-12 strain sources, DH5 α for example, JM109, preferred E.coli HB101.Under the effect that helps bacterium, the joint efficiency of donor bacterium and recipient bacterium strengthens greatly.
Among the present invention, the mgdh gene knockout bacteria growing of gained and utilize glucose slower preferably also comprises 3 for this reason) step of the adaptive evolution of oxidizing glucose acidfast bacilli mgdh clpp gene degerming.Specifically: cultivate in glucose is the substratum of sole carbon source, each fixes time switching once, up to obtaining thalline yield and the tangible bacterial strain of specific growth rate.Transferred once in preferred per 24 hours, inoculative proportion is 1: 100 (volume ratio), cultured continuously 10 days.Described glucose be the composition of substratum of sole carbon source preferable contain glucose and yeast powder, better glucose 10g/L, the yeast powder 20g/L of containing.
The present invention solves the problems of the technologies described above four of the technical scheme that adopted: the purposes of aforesaid oxidizing glucose acidfast bacilli mgdh clpp gene degerming.As the biological catalyst in biological industry and the synthetic field of chirality.Be applied to the industrial production of vitamins C, otan, miglitol especially.
Raw material that the present invention is used or reagent except that specifying, all commercially available getting.
Than prior art, beneficial effect of the present invention is as follows: the present invention adopts gene knockout and the resulting Gluconobater oxydans genetic engineering strain GDHE of adaptive evolution, can adopt cheap relatively glucose to cultivate as carbon source, obtain higher growth velocity and thalline yield, the interpolation concentration of glucose will be added concentration 80g/L well below the sorbyl alcohol of industrial application simultaneously, only is 10g/L.Therefore be not that the catalyzed reaction of key enzyme is as in the industrial production of vitamin C precursor, anti-type ii diabetes medicine miglitol precursor and otan etc. in conjunction with Hexose phosphate dehydrogenase for industrial application with film, can replace sorbyl alcohol etc. as fermenting carbon source with glucose fully, carry out cultivating before the transformation, can save production cost greatly.
Description of drawings
Below in conjunction with description of drawings feature of the present invention and beneficial effect.
Fig. 1 is a knockout carrier building process synoptic diagram.
Fig. 2 is a homology double exchange schematic flow sheet.
Fig. 3 knocks out bacterium in the screening that contains on the glucose yeast powder solid plate of lime carbonate, and the oxidizing glucose acidfast bacilli that successfully knocks out the mgdh gene does not produce transparent circle on this flat board, and the success still can produce transparent circle.
Fig. 4 is oxidizing glucose acidfast bacilli 621H, clpp gene degerming GDHK and the growing state of evolution bacterium GDHE in glucose yeast powder liquid substratum.Wherein, 621H (△), GDHK (◆), GDHE ().
Embodiment
Because cause the oxidizing glucose acidfast bacilli on glucose, grow the reason of catalytic performance difference mainly be because film in conjunction with the oxidation of Hexose phosphate dehydrogenase to glucose, the gluconic acid that generates causes the reduction that has of pH in culture medium, has finally influenced the growth and the catalytic performance of thalline.The present invention has at first made up mgdh clpp gene degerming GDHK, eliminate film causes acid in conjunction with Hexose phosphate dehydrogenase catalysis accumulation, the method of evolving by metabolism has obtained being suitable for the bacterial strain GDHE with higher thalline yield and better growth characteristics that industrial production is used simultaneously.
Below in conjunction with specific embodiment, the invention will be further described.Should be understood that following examples only are used to the present invention is described but not are used to limit scope of the present invention.
The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, the condition described in " molecular cloning: laboratory manual " is carried out.
The structure of embodiment 1 knock-out bacterial strain GDHK (/ Δ mgdh)
Utilize the method for homology exchange to carry out film knocking out in the oxidizing glucose acidfast bacilli genome in conjunction with Hexose phosphate dehydrogenase (mgdh) gene.The mgdh gene is responsible for the reaction that the catalysis glucose oxidase generates gluconic acid on film, be substratum acidifying major cause.Specific as follows:
1.1 the structure of knockout carrier pSUP202-mgdh::Gm
Extracting the complete genome DNA of oxidizing glucose acidfast bacilli 621H (buy from German DSMZ limited-liability company), is masterplate with it, utilizes following primer to distinguishing pcr amplification upstream fragment gdh1 and downstream fragment gdh2.
Primer is to 1:
gdh?1F-Nco?Ⅰ：AAT
CCATGG?GGCTGCCCTCTACCTGTT；
gdh?1R-Kpn?Ⅰ-Xba?Ⅰ-EcoR?Ⅰ：GCC
GAATTC?
TCTAGA?
GGTACC?GCATCGAACACCCAGACA。
Primer is to 2:
gdh2F-Xba?Ⅰ：AAC
TCTAGA?AGCCACCCTGTCTTCCAC；
gdh2R-EcoR?Ⅰ：AAC
TCTAGA?AGCCACCCTGTCTTCCAC。
With plasmid pBBR1MCS-5 (Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM, and Peterson KM.Four new derivatives of the broad-hostrange cloning vector pBBR1MCS carrying different antibioticresistance cassettes.Gene.1995,166:175-176. taking from the upright University Medical Center microbiology of Louisiana, United States is .Kovach ME professor with immunology) be masterplate, with the following primer PCR gentamicin resistant gene (Gm) that increases:
GmF-Kpn?Ⅰ：TAA
GGTACC?CGTGGAAACGGATGAAGG；
GmR-Xba?Ⅰ：GCG
TCTAGA?TCTCGGCTTGAACGAATT。
Reclaim the PCR product, enzyme is cut, be connected to carrier pSUP202 (Simon R as shown in Figure 1, Priefer U, Puhler A.A broad host range mobilization system in vivo genetic engineering:transposon mutagenesis in Gram negative bacteria.Biotechnology.1983,1:784-791. taking from biological institute of German University Bielefeld genetics is .Simon R professor) on, thereby obtain recombinant plasmid pSUP202-mgdh::Gm, finish the structure of knockout carrier.Empirical tests, contained sequence is to be that the Genbank accession number is that the 180th～1151 of AAW60048, accession number are the 3575th～4608, the 1186th～2231 of U25061.1 successively in the knockout carrier.
1.2 knockout carrier imported in the oxidizing glucose acidfast bacilli born of the same parents finishes gene knockout
1. the foundation of donor bacterium:, obtain containing the E.coli JM109 of recombinant plasmid pSUP202-mgdh::Gm with recombinant plasmid pSUP202-mgdh::Gm transformed competence colibacillus E.coliJM109 (purchase) from novagen company.
Help the foundation of bacterium:, obtain containing the E.coli HB101 of helpful plasmid pRK2013 with plasmid pRK2013 (purchasing company) transformed competence colibacillus E.coliHB101 (purchase) from novagen company in Clontech.
2. yeast culture: respectively with the donor bacterium, help bacterium to be seeded among the LB that respectively is added with gentamicin 100mg/L and kantlex 100mg/L to cultivate about 8 hours.Recipient bacterium oxidizing glucose acidfast bacilli 621H is seeded in the sorbyl alcohol substratum (sorbyl alcohol 80g/L, yeast powder 20g/L, Cp 00mg/L) and cultivated about 18 hours.
3. three parents engage: get above-mentioned receptor parent bacterium liquid 1.5ml, centrifugal collection thalline is outwelled supernatant, wash twice with physiological saline after, outwell liquid, add 0.5ml and help bacterium liquid, add 1.5ml donor bacterium behind the centrifugal collection thalline, wash once with the sorbyl alcohol substratum behind the centrifugal collection thalline, outwell liquid, in remaining a small amount of substratum with micropipet with the cell mixing, transfer on the Neosorb substratum of added with antibiotic not and be coated with, be inverted 30 ℃ of overnight incubation.
Recombinant plasmid pSUP202-mgdh::Gm can enter oxidizing glucose acidfast bacilli 621H under the help that helps bacterium, by having the homologous fragment (gdh1 of film thereon in conjunction with Hexose phosphate dehydrogenase, gdh2), therefore can with the mgdh dna fragmentation generation double exchange on the oxidizing glucose acidfast bacilli 621H karyomit(e), the mgdh fragment that will contain the Gm gene is incorporated on the karyomit(e) of oxidizing glucose acidfast bacilli 621H, thereby makes film in conjunction with the glucose dehydrogenase gene inactivation.The schematic flow sheet of homology double exchange is seen Fig. 2.
4. zygosporic screening: three close zygomycetes body and function aseptic double-distilled waters of overnight incubation are washed from the Neosorb substratum, be applied on the Neosorb culture medium flat plate that adds cynnematin and each 100mg/L of gentamicin, cultivated 2～4 days.Oxidizing glucose acidfast bacilli 621H itself has cynnematin (Cefoxitin) resistance, and Cefoxitin is used for killing the intestinal bacteria that three parent's cohesive process have the gentamicin resistance.Plasmid pSUP202 is a suicide plasmid, soon just can not exist after entering oxidizing glucose acidfast bacilli 621H, the mgdh fragment that only contains the Gm resistant gene is incorporated into the oxidizing glucose acidfast bacilli 621H tool Gm resistance just on the karyomit(e), therefore screens three close zygotes by Cefoxitin, Gm.
1.3 knock out screening and the checking of bacterium
The zygote that screens further is transferred on the glucose solids substratum (composition: glucose 20g/L, yeast powder 20g/L, lime carbonate 10g/L) that contains lime carbonate with replica-plating method.Cultivated 2-4 days for 30 ℃.Knock out son because lack film,, can not produce transparent circle, show as Fig. 3 in its periphery of bacterial colonies so on dextrose culture-medium, can not produce acid in conjunction with Hexose phosphate dehydrogenase.According to this principle, remove the false positive bacterium colony that obtains in the primary dcreening operation, the bacterium colony that does not have transparent circle around the picking promptly obtains knocking out bacterium GDHK.
The evolution of embodiment 2 oxidizing glucose acidfast bacilli mgdh clpp gene degerming
Place the 50ml substratum in the bottle shaking of 250ml, medium component is: glucose 10g/l and yeast powder 20g/l.Do not add any microbiotic in the substratum.Inoculative proportion is 1: 100 (volume ratio), and switching in about 24 hours is (change with the thalline doubling time in the culturing process changes initial inoculation amount or transit time) once.Thalline yield and the specific growth rate of cultivating the 10th day bacterial strain are significantly improved, called after GDHE.
With original bacterium G.oxydans 621H with knock out mutant GDHK, GDHE and in the sorbyl alcohol substratum, activate OD600=1.0, inoculum size with 1% is transferred to the fresh dextrose culture-medium of 50ml (composition: glucose 10g/L, yeast powder 20g/L) in, 30 ℃ of shaking tables are cultivated, OD600 value of survey in per 1 hour.Contrast OD value and dry weight standard curve determine that the dry weight of thalline correspondence changes, and draw growth curve, see Fig. 4, and X-coordinate is to change the time, and ordinate zou is corresponding dry weight.As seen, by after knocking out and evolving, the more original bacterium of cell stand density of GDHE has improved 163%.
In sum, adopt gene knockout and the resulting Gluconobater oxydans genetic engineering strain GDHE of adaptive evolution, can adopt cheap relatively glucose to cultivate as carbon source, and can obtain higher growth velocity and thalline yield, the interpolation concentration of glucose will be added concentration 80g/L well below the sorbyl alcohol of industrial application simultaneously, only is 10g/L.Therefore be not that the catalyzed reaction of key enzyme is as in the industrial production of vitamin C precursor, anti-type ii diabetes medicine miglitol precursor and otan etc. in conjunction with Hexose phosphate dehydrogenase for industrial application with film, can replace sorbyl alcohol etc. as fermenting carbon source with glucose fully, carry out cultivating before the transformation, can save production cost greatly.
Claims (10)
1. a recombinant vectors that is used for gene knockout is characterized in that, it contains oxidizing glucose acidfast bacilli (Gluconobacter oxydans) mgdh dna homolog reorganization forearm, marker gene and mgdh dna homolog reorganization postbrachium successively.
2. recombinant vectors as claimed in claim 1 is characterized in that described marker gene is an antibiotics resistance gene.
3. recombinant vectors as claimed in claim 1 is characterized in that, the length of described mgdh dna homolog reorganization forearm and postbrachium is 0.5～1.5kb.
4. recombinant vectors as claimed in claim 1 is characterized in that, described recombinant vectors is plasmid pSUP202.
5. the clpp gene degerming of an oxidizing glucose acidfast bacilli is characterized in that, it is the degerming of mgdh clpp gene.
6. clpp gene degerming as claimed in claim 5 is characterized in that, described oxidizing glucose acidfast bacilli is oxidizing glucose acidfast bacilli 621H.
7. the preparation method as claim 5 or 6 described oxidizing glucose acidfast bacilli clpp gene degerming is characterized in that, may further comprise the steps:
1) with each described recombinant vectors transformed into escherichia coli that is used for gene knockout of claim 1～4, obtains transformant;
2) with the transformant of step 1) gained as the donor bacterium, oxidizing glucose acidfast bacilli action receptor bacterium, carrying out two parents engages, described recombinant vectors enters neutralize the mgdh gene generation homology double exchange on its karyomit(e) of oxidizing glucose acidfast bacilli, and the zygote of acquisition is the degerming of mgdh clpp gene.
8. preparation method as claimed in claim 7 is characterized in that step 2) in also add to help bacterium, carry out three parents and engage, described help bacterium is to contain the intestinal bacteria that help plasmid pRK2013 or PRK2073.
9. as claim 7 or 8 described preparation methods, it is characterized in that, also comprise 3) step of the adaptive evolution of oxidizing glucose acidfast bacilli mgdh clpp gene degerming: in being the substratum of sole carbon source, cultivates glucose, each fixes time switching once, up to obtaining thalline yield and the tangible bacterial strain of specific growth rate.
10. as the purposes of claim 5 or 6 each described oxidizing glucose acidfast bacilli mgdh clpp gene degerming, it is characterized in that, as the biological catalyst in biological industry and the synthetic field of chirality.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN106282218A (en) * | 2016-09-20 | 2017-01-04 | 黑龙江省科学院微生物研究所 | A kind of method of nif gene nifDK gene knockout |
| CN109370972A (en) * | 2018-11-19 | 2019-02-22 | 江南大学 | A kind of acetobacter engineering bacteria and its application |
| CN109402033A (en) * | 2017-10-31 | 2019-03-01 | 天津科技大学 | A kind of efficient hydrogenlike silicon ion engineering bacteria and its construction method using glucose |
| CN109456924A (en) * | 2018-12-20 | 2019-03-12 | 河南大学 | A method of increase and not holds in the palm gluconobacter sp HD924 biomass |
| CN109628367A (en) * | 2019-01-30 | 2019-04-16 | 江南大学 | A method of improving Gluconobacter oxvdans sorb candy output and production intensity |
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2010
- 2010-07-16 CN CN 201010228337 patent/CN102146415A/en active Pending
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| 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 20100507 Vera Krajewski等 Metabolic Engineering of Gluconobacter oxydans for Improved Growth Rate and Growth Yield on Glucose by Elimination of Gluconate Formation 第4369-4376页 1-10 第76卷, 第13期 * |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN106282218A (en) * | 2016-09-20 | 2017-01-04 | 黑龙江省科学院微生物研究所 | A kind of method of nif gene nifDK gene knockout |
| CN109402033A (en) * | 2017-10-31 | 2019-03-01 | 天津科技大学 | A kind of efficient hydrogenlike silicon ion engineering bacteria and its construction method using glucose |
| CN109370972A (en) * | 2018-11-19 | 2019-02-22 | 江南大学 | A kind of acetobacter engineering bacteria and its application |
| CN109456924A (en) * | 2018-12-20 | 2019-03-12 | 河南大学 | A method of increase and not holds in the palm gluconobacter sp HD924 biomass |
| CN109628367A (en) * | 2019-01-30 | 2019-04-16 | 江南大学 | A method of improving Gluconobacter oxvdans sorb candy output and production intensity |
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Application publication date: 20110810 |