CN103710375A - Novel plasmid for gene modification of Corynebacterium glutamicum and application thereof - Google Patents
Novel plasmid for gene modification of Corynebacterium glutamicum and application thereof Download PDFInfo
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- CN103710375A CN103710375A CN201310742881.XA CN201310742881A CN103710375A CN 103710375 A CN103710375 A CN 103710375A CN 201310742881 A CN201310742881 A CN 201310742881A CN 103710375 A CN103710375 A CN 103710375A
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Abstract
The invention discloses a novel plasmid which simultaneously realizes gene knockout and over-expression in a Corynebacterium glutamicum genome and carries no resistance maker, belonging to the field of gene engineering. The novel plasmid disclosed by the invention can simultaneously realize knockout of a gene and over-expression of another gene in a Corynebacterium glutamicum genome, and carries no resistance marker; and the over-expression gene inserted in the Corynebacterium glutamicum genome can be stably inherited and expressed in a host. On the other hand, a target recombinant strain is obtained through the method by adopting double-exchange homologous recombination principles, so that the defect of low recombination efficiency in a single-exchange homologous recombination process is overcome, thus greatly increasing the probability of obtaining the target recombinant strain. The provision of the method is beneficial to gene engineering modification of Corynebacterium glutamicum and reduces the influence caused by introduction of resistance marker genes.
Description
Technical field
The present invention relates to a kind of novel plasmid and application thereof that realizes gene knockout and cross expression in Corynebacterium glutamicum gene group, especially a kind of gene knockout of simultaneously realizing by once recombinating is not with any resistance marker with crossing expression, belong to gene engineering technology field.
Background technology
Corynebacterium glutamicum (Corynebacterium glutamicum) is the isolated non-bacterium of curing the disease from soil.This bacterium has vital role economically, be widely used in industrial fermentation and produced each seed amino acid, as Pidolidone and 1B (Georgi etc., 2005, Lysine and glutamate production by Corynebacterium glutamicum on glucose, fructose and sucrose:Roles of malic enzyme and fructose-1, 6-bisphosphatase.Metab.Eng.7 (4): 291-301 and Becker etc., 2011, From zero to hero-design-based systems metabolic engineering of Corynebacterium glutamicum for L-lysine production.Metab.Eng.13 (2): 159-168.).Along with directly acting on development and the complete genomic parsing of Corynebacterium glutamicum of DNA technique, utilize genetic engineering means improvement Corynebacterium glutamicum to become possibility, and replace gradually classical random mutation breeding (Tauch etc., 2003, Plasmids in Corynebacterium glutamicum and their molecular classification by comparative genomics.J.Biotechnol.104 (1-3): 27-40
.
Utilizing crossing of key enzyme gene in the amino acid route of synthesis of expression plasmid mediation to express is Main Means (Ikeda and the Katsumata that at present Corynebacterium glutamicum is carried out to genetic modification, 1998, A novel system with positive selection for the chromosomal integration of replicative plasmid DNA in Corynebacterium glutamicum.Microbiol.144 (Pt7): 1863-1868).Yet, utilize crossing of expression plasmid mediated gene to express and will in Corynebacterium glutamicum cell, introduce resistant gene (as ampicillin resistance gene or kalamycin resistance gene etc.), in order to maintain the stable of plasmid, in fermenting process, need to add certain density microbiotic in substratum.In fermenting process, add microbiotic and have some unfavorable factors: (1) increase cost; (2) change the physiological function of Corynebacterium glutamicum; (3) increase people to the panic (Tauch etc. of microbiotic, 2002, The alanine racemase gene alr is an alternative to antibiotic resistance genes in cloning systems for industrial Corynebacterium glutamicum strains.J.Biotechnol.99 (1): 79-91; Zhou etc., 2009, A reusable method for construction of non-marker large fragment deletion yeast auxotroph strains:A practice in Torulopsis glabrata.J.Microbial.Meth.76 (1): 70-74).Therefore, remove crossing of expression plasmid mediated gene and express, goal gene is inserted into and in genome, crosses that to express be the main trend that following Corynebacterium glutamicum utilizes genetic engineering means transformation.
Contain the non-replicating plasmid of DNA fragmentation and resistant gene in a fragment gene group and be fabricated and be successfully used in (Correia etc. in Corynebacterium glutamicum strain improvement, 1996, Targeted integration of foreign genes into repetitive sequences of the Brevibacterium lactofermentum genome.FEMS Microbiol.Lett.142 (2-3): 259-264 and Amador etc., 2000, A Brevibacterium lactofermentum16S rRNA gene used as target site for homologous recombination.FEMS Microbiol.Lett.185 (2): 199-204).The goal gene that this non-replicating plasmid can be expressed the need that carry is inserted in genome, and does not need the mediation of expression plasmid.Yet the acquisition of object bacterial strain is to change homologous recombination by single cross, this recombination method obtains probability low (Ikeda and the Katsumata of object bacterial strain, 1998, A novel system with positive selection for the chromosomal integration of replicative plasmid DNA in Corynebacterium glutamicum.Microbiol.144 (Pt7): 1863-1868).On the other hand, the object bacterial strain obtaining also carries resistant gene.
In the present invention, we have built novel non-replicating pK18mobsacB-Δ A::B, this plasmid contains DNA fragmentation in the genome of two ends, and electricity forwards in Corynebacterium glutamicum cell and obtains object bacterial classification by double exchange homologous recombination, thereby greatly improves object bacterial classification yield.On the other hand, utilize object bacterial classification that this plasmid obtains without how antibiotic marker gene.As application example, we have successfully realized knocking out with crossing of lysC gene of pck gene and have expressed in Corynebacterium glutamicum.
Summary of the invention
The object of the invention is to build a kind of novel non-replicating plasmid, this plasmid electricity forwards to can realize gene knockout and gene overexpression and without any resistance marker simultaneously in Corynebacterium glutamicum.
Technical scheme of the present invention: it is example that a kind of novel non-replicating plasmid pK18mobsacB-Δ A::B(be take non-replicating plasmid pK18mobsacB-Δ pck::lysC).
Contain kalamycin resistance gene kan;
Contain sucrose lethal gene sacB;
The one section of sequence in upstream (1212bp) that contains pck gene in Corynebacterium glutamicum gene group and the one section of sequence in downstream (1220bp);
Contain Ptac-lysC-rrnBT1T2 expression cassette, this expression cassette is by a tac promotor in intestinal bacteria and excellent bacillus with transcripting starting function, SD sequence from lysC gene in Corynebacterium glutamicum, from lysC operon in Corynebacterium glutamicum gene group, the rrnBT1T2 in intestinal bacteria and excellent bacillus with Transcription Termination function forms.
It is example that novel non-replicating plasmid pK18mobsacB-Δ A::B(be take non-replicating plasmid pK18mobsacB-Δ pck::lysC) construction process:
The first step, the Corynebacterium glutamicum ATCC13032 genome of take is template, pck-L-F and pck-L-R are template, react the pck-L left arm fragment of amplification 1212bp by PCR, at 5 ' and 3 ' end of PCR product, introduce respectively HindIII and SalI restriction enzyme site.With restriction enzyme HindIII and SalI respectively enzyme cut pck-L fragment and non-replicating plasmid pK18mobsacB, after purifying, both enzymes are connected, obtaining thus plasmid size is another non-replicating plasmid of 6931bp, called after pK18mobsacB-pck-L;
Second step, the Corynebacterium glutamicumATCC13032 genome of take is template, pck-R-F and pck-R-R are template, react the pck-R right arm fragment of amplification 1220bp by PCR, at 5 ' and 3 ' end of PCR product, introduce respectively SalI and XbaI restriction enzyme site.With restriction enzyme SalI and XbaI respectively enzyme cut pck-R fragment and non-replicating plasmid pK18mobsacB-pck-L, after purifying, both enzymes are connected, obtaining thus plasmid size is another non-replicating plasmid of 8151bp, called after pK18mobsacB-Δ pck;
The 3rd step, the Corynebacterium glutamicum ATCC13032 genome of take is template, lysC-F and lysC-R are template (note: contain Corynebacterium glutamicum SD sequence in lysC-F sequence), by PCR, react the lysC operon of amplification 1226bp, at 5 ' and 3 ' end of PCR product, introduce respectively EcoRI and NheI restriction enzyme site.With restriction enzyme EcoRI and NheI respectively enzyme cut lysC operon and expressive plasmid pDXW-8, after purifying, both enzymes are connected, obtaining thus plasmid size is another expressive plasmid of 10774bp, called after pDXW-8-lysC;
Finally, take expressive plasmid pDXW-8-lysC as template, Ptac-F and Ptac-R are template, react the Ptac-lysC-rrnBT1T2 expression cassette of amplification 1226bp by PCR, at 5 ' and 3 ' end of PCR product, introduce SalI restriction enzyme site.With restriction enzyme SalI respectively enzyme cut Ptac-lysC-rrnBT1T2 expression cassette and non-replicating plasmid pK18mobsacB-Δ pck, with alkaline phosphatase CIAP dephosphorylation, after purifying, both enzymes are connected, obtaining thus plasmid size is another non-replicating plasmid of 10098bp, called after pK18mobsacB-Δ pck::lysC.
Obtain the method (bacterial strain that obtains pck gene knockout and lysC gene overexpression of take is example) of object bacterial strain:
The first step, non-replicating plasmid pK18mobsacB-Δ pck::lysC transforms and enters in cell;
Second step, by homologous recombination (being pck-L homologous recombination in pck-L on non-replicating plasmid and Corynebacterium glutamicum gene group), make non-replicating Plasmids conjugation in Corynebacterium glutamicum gene group, screening is at the dull and stereotyped bacterial strain that can normal growth of kalamycin resistance;
The 3rd step, select the bacterial strain of a strain on kalamycin resistance flat board and cultivate 24h in not containing antibiotic LBG liquid nutrient medium, make this non-replicating plasmid pK18mobsacB-Δ pck::lysC and Corynebacterium glutamicum gene group that homologous recombination for the second time (being pck-R homologous recombination in pck-R on non-replicating plasmid and genome) occur, remove the kalamycin resistance gene in genome, the bacterial strain that screening is grown on the LB flat board that contains 10% sucrose concentration.
The 4th step, utilize primer Ptac-F and Ptac-R to carry out bacterium colony PCR to the bacterium colony on the LB flat board of 10% sucrose concentration, to determine that non-replicating plasmid pK18mobsacB-Δ pck::lysC and Corynebacterium glutamicum gene group, at pck gene locus, double exchange homologous recombination successfully occurs.
Finally, select object recombinant bacterial strain, in 30 generations of cultured continuously in the LBG substratum that does not contain any selection markers (as the sucrose mark of kantlex or 10%), measure the enzyme of corresponding gene coding and live.The little recombinant bacterial strain of enzyme variation alive is the stable object recombinant bacterial strain of heritability.
Described novel plasmid is equally applicable to the Gram-negative of sucrose and kalamycin resistance sensitivity or the application in positive strain.
The method disclosed in the present, can in Corynebacterium glutamicum gene group, in the same time, realize crossing of gene knockout and another gene to express and without any resistance marker, be inserted in the heredity that gene that crossing in Corynebacterium glutamicum gene group express can be stable in host and express.The method uses double exchange homologous recombination principle to obtain object recombinant bacterial strain, and this has overcome the shortcoming that low recombination efficiency in homologous recombination process is changed in single cross, thereby greatly improves the probability that obtains object recombinant bacterial strain.The object bacterial strain that the method obtains does not contain any resistance marker, can realize crossing of a gene knockout and another gene simultaneously and express.
Accompanying drawing explanation
The building process figure (E1 in figure, E2 and E3 represent respectively different restriction enzymes) of Fig. 1 non-replicating plasmid pK18mobsacB-Δ A::B.
Fig. 2 carries out genetic manipulation procedure chart to Corynebacterium glutamicum gene group.
Embodiment
The structure of embodiment 1 non-replicating plasmid pK18mobsacB-Δ A::B (the non-replicating plasmid pK18mobsacB-Δ pck::lysC of take is example)
The structure of non-replicating plasmid pK18mobsacB-Δ A::B as shown in Figure 1.The Corynebacterium glutamicum ATCC13032 genome of take is template, pck-L-F and pck-L-R are template, by PCR, react the pck-L fragment of amplification 1212bp, at 5 ' and 3 ' end of PCR product, introduce respectively HindIII and SalI restriction enzyme site.With restriction enzyme HindIII and SalI respectively enzyme cut pck-L fragment and non-replicating plasmid pK18mobsacB, after purifying, both enzymes are connected, obtaining thus plasmid size is another non-replicating plasmid of 6931bp, called after pK18mobsacB-pck-L;
The Corynebacterium glutamicum ATCC13032 genome of take is template, pck-R-F and pck-R-R are template, by PCR, react the pck-R fragment of amplification 1220bp, at 5 ' and 3 ' end of PCR product, introduce respectively SalI and XbaI restriction enzyme site.With restriction enzyme SalI and XbaI respectively enzyme cut pck-R fragment and non-replicating plasmid pK18mobsacB-pck-L, after purifying, both enzymes are connected, obtaining thus plasmid size is another non-replicating plasmid of 8151bp, called after pK18mobsacB-Δ pck;
The Corynebacterium glutamicum ATCC13032 genome of take is template, lysC-F and lysC-R are template (note: contain SD sequence in lysC-F sequence), by PCR, react the lysC operon of amplification 1226bp, at 5 ' and 3 ' end of PCR product, introduce respectively EcoRI and NheI restriction enzyme site.With restriction enzyme EcoRI and NheI respectively enzyme cut lysC operon and expressive plasmid pDXW-8, after purifying, both enzymes are connected, obtaining thus plasmid size is another expressive plasmid of 10774bp, called after pDXW-8-lysC;
Take expressive plasmid pDXW-8-lysC as template, and Ptac-F and Ptac-R are template, react the Ptac-lysC-rrnBT1T2 expression cassette of amplification 1226bp by PCR, at 5 ' and 3 ' end of PCR product, introduce SalI restriction enzyme site.With restriction enzyme SalI respectively enzyme cut Ptac-lysC-rrnBT1T2 expression cassette and non-replicating plasmid pK18mobsacB-Δ pck, with alkaline phosphatase CIAP dephosphorylation, after purifying, both enzymes are connected, obtaining thus plasmid size is another non-replicating plasmid of 10098bp, called after pK18mobsacB-Δ pck::lysC.
The primer sequence using in building process is as table 1, and PCR reaction conditions is as table 2.
Table 1.PCR increases, and (underscore is restriction enzyme site to required primer sequence; Bold Italic is SD sequence)
PCR relevant information in table 2. the present invention
Embodiment 2 obtains the method (bacterial strain that obtains pck gene knockout and lyC gene overexpression of take is example) of object bacterial strains:
Non-replicating plasmid pK18mobsacB-Δ pck::lysC is transformed and entered in cell.By homologous recombination (being pck-L homologous recombination in pck-L on non-replicating plasmid and Corynebacterium glutamicum gene group), make non-replicating Plasmids conjugation in Corynebacterium glutamicum gene group, screening is at the dull and stereotyped bacterial strain that can normal growth of kalamycin resistance.Select the bacterial strain of a strain on kalamycin resistance flat board and do not containing antibiotic LBG(10gL
-1peptone, 5gL
-1yeast powder, 10gL
-1naCl, 5gL
-1glucose) in liquid nutrient medium, cultivate 24h, make this non-replicating plasmid pK18mobsacB-Δ pck::lysC and Corynebacterium glutamicum gene group that homologous recombination for the second time (being pck-R homologous recombination in pck-R on non-replicating plasmid and genome) occur, remove the kalamycin resistance gene in genome, the bacterial strain that screening is grown on the LB flat board that contains 10% sucrose concentration.Utilize primer Ptac-F and Ptac-R to carry out bacterium colony PCR to the bacterium colony on the LB flat board of 10% sucrose concentration, to determine that non-replicating plasmid pK18mobsacB-Δ pck::lysC and Corynebacterium glutamicum gene group, at pck gene locus, double exchange homologous recombination successfully occurs.
Select object recombinant bacterial strain, in 30 generations of cultured continuously in the LBG substratum that does not contain any selection markers (as the sucrose mark of kantlex or 10%), measure the enzyme of corresponding gene coding and live.The little recombinant bacterial strain of enzyme variation alive is the stable object recombinant bacterial strain of heritability.
The above method is not only applicable to Corynebacterium glutamicum, other any Gram-negative or positive strain to sucrose and kalamycin resistance sensitivity is all suitable for, so these contents all belongs to protection scope of the present invention.
Claims (9)
1. for a novel plasmid for Corynebacterium glutamicum gene transformation, this non-replicating plasmid is pK18mobsacB-Δ pck::lysC, comprises following feature:
1) contain kalamycin resistance gene kan and sucrose lethal gene sacB;
2) contain one section by one section of upstream sequence of pck gene and one section of disappearance Δ pck gene fragment that downstream sequence forms;
3) contain a Ptac-lysC-rrnBT1T2 expression cassette.
2. novel plasmid according to claim 1, is characterized in that the plasmid that sets out of described non-replicating plasmid is pK18mobsacB.
3. novel plasmid according to claim 1, it is characterized in that described Ptac-lysC-rrnBT1T2 expression cassette is by a tac promotor in intestinal bacteria and excellent bacillus with transcripting starting function, SD sequence from Corynebacterium glutamicum gene, from lysC operon in Corynebacterium glutamicum gene group, the rrnBT1T2 in intestinal bacteria and excellent bacillus with Transcription Termination function forms.
4. the application method of novel plasmid according to claim 1 in Corynebacterium glutamicum, is characterized in that comprising the steps:
1) plasmid construction is a). and Corynebacterium glutamicum (Corynebacterium glutamicum) the ATCC13032 genome of take is template, pck-L-F and pck-L-R are primer, by PCR, react amplification pck-L left arm fragment, 5 ' and the 3 ' end at PCR product is introduced respectively restriction enzyme site, with restriction enzyme, builds non-replicating plasmid pK18mobsacB-pck-L; B). the Corynebacterium glutamicum ATCC13032 genome of take is template, and pck-R-F and pck-R-R are primer, by PCR, reacts amplification pck-R right arm fragment, at 5 ' and 3 ' end of PCR product, introduces respectively restriction enzyme site.With restriction enzyme S respectively enzyme cut pck-R fragment and non-replicating plasmid pK18mobsacB-pck-L, after purifying, both enzymes are connected and build non-replicating plasmid pK18mobsacB-Δ pck; C). the Corynebacterium glutamicum ATCC13032 genome of take is template, and lysC-F and lysC-R are primer, construction expression type plasmid pDXW-8-lysC; D). take expressive plasmid pDXW-8-lysC as template, Ptac-F and Ptac-R are template, PCR reaction amplification Ptac-lysC-rrnBT1T2 expression cassette, is inserted in non-replicating plasmid pK18mobsacB-Δ pck, builds non-replicating plasmid pK18mobsacB-Δ pck::lysC;
2) homologous recombination forwards non-replicating plasmid pK18mobsacB-Δ pck::lysC electricity in Corynebacterium glutamicum competent cell to for the first time, then kalamycin resistance plate screening positive colony bacterial strain;
3) homologous recombination is not being cultivated 24h containing in antibiotic LBG liquid nutrient medium for the second time, there is homologous recombination for the second time in the pK18mobsacB-Δ pck::lysC plasmid sequence self that makes to be incorporated in Corynebacterium glutamicum, knock out kalamycin resistance gene, then screening positive clone bacterial strain on the LB flat board that contains 10% sucrose concentration.
5. application method according to claim 4, is characterized in that the SD sequence that contains Corynebacterium glutamicum gene in lysC-F sequence in described step 1).
6. application method according to claim 4, is characterized in that in described step 1) that Δ pck gene fragment in non-replicating plasmid is the sequence that contains pck gene left and right two arms in Corynebacterium glutamicum and the gene fragment of intermediate sequence disappearance.
7. application method according to claim 4, it is characterized in that described step 2) in non-replicating plasmid be by the homologous sequence generation homologous recombination of pck gene in left arm on Δ pck gene fragment gene or right arm and Corynebacterium glutamicum gene group, make non-replicating plasmid integration in Corynebacterium glutamicum gene group.
8. application method according to claim 4, is characterized in that in described step 3) that homologous recombination is for the second time that homologous sequence by being incorporated in the Δ pck gene fragment in Corynebacterium glutamicum in a left arm or right arm and genome is recombinated.
9. novel plasmid according to claim 1 is to the Gram-negative of sucrose and kalamycin resistance sensitivity or the application in positive strain.
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| CN109294966A (en) * | 2018-10-26 | 2019-02-01 | 江南大学 | A kind of the Corynebacterium glutamicum recombinant bacterium and its construction method of high yield L-Leu |
| CN111073841A (en) * | 2019-11-25 | 2020-04-28 | 华农（肇庆）生物产业技术研究院有限公司 | Corynebacterium ATCC13032 improved strain capable of effectively expressing foreign protein and construction method thereof |
| CN111893146A (en) * | 2020-08-26 | 2020-11-06 | 无锡晶扬生物科技有限公司 | Culture medium for improving yield of corynebacterium glutamicum tetrahydropyrimidine and fermentation method |
| CN114480467A (en) * | 2022-02-24 | 2022-05-13 | 江南大学 | CRISPR-cpf1 screening tool for assisting sacB gene editing system in corynebacterium |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109294966A (en) * | 2018-10-26 | 2019-02-01 | 江南大学 | A kind of the Corynebacterium glutamicum recombinant bacterium and its construction method of high yield L-Leu |
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| CN111893146A (en) * | 2020-08-26 | 2020-11-06 | 无锡晶扬生物科技有限公司 | Culture medium for improving yield of corynebacterium glutamicum tetrahydropyrimidine and fermentation method |
| CN114480467A (en) * | 2022-02-24 | 2022-05-13 | 江南大学 | CRISPR-cpf1 screening tool for assisting sacB gene editing system in corynebacterium |
| CN114480467B (en) * | 2022-02-24 | 2023-08-25 | 江南大学 | CRISPR-cpf1 screening tool for assisting sacB gene editing system in coryneform bacteria |
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Application publication date: 20140409 |