CN106047916A - Corynebacterium glutamicum strain for production of 5-aminolevulinic acid and construction and application of corynebacterium glutamicum strain - Google Patents
Corynebacterium glutamicum strain for production of 5-aminolevulinic acid and construction and application of corynebacterium glutamicum strain Download PDFInfo
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Abstract
The invention discloses a corynebacterium glutamicum strain for production of 5-aminolevulinic acid and construction and application of corynebacterium glutamicum strain. A construction method includes: (1) deleting a lactic dehydrogenase coding gene 1dhA and acetic acid generation genes pta-ackA, pqo and cat in corynebacterium glutamicum to obtain a strain named CB4; inserting a strong sod promoter in front of a phosphoenolpyruvate carboxylase coding gene ppc in the strain CB4 to obtain a strain CB5; deleting a gene pck in the strain CB5 to obtain a strain CB6; (2) transferring plasmid pXA and plasmid pEP2<tuf>-rhtA into the strain CB6. The strain constructed according to the method is capable of generating 2.78g/L 5-aminolevulinic acid in a culture medium with 10g/L glucose serving as a carbon source, which lays a foundation for subsequent continuous feeding of a fermentation tank to increase yield of the 5-aminolevulinic acid.
Description
Technical field
The invention belongs to biotechnology and application, more particularly to a kind of paddy ammonia producing 5-ALA
Acid coryneform bacterial strains and structure and application.
Background technology
5-ALA, molecular weight is 131.13, and fusing point is 118 DEG C.It it is a kind of non-protein amino acid.5-amino second
Acyl propanoic acid has that side effect is little, good penetrability feature, be widely used in skin carcinoma, bladder cancer, digestive tract cancer,
In the diagnosis of pulmonary carcinoma and optical dynamic therapy (PDT).Meanwhile, 5-ALA is the most also having critically important application such as
As plant growth regulator, herbicide and environment friendly agricultural etc..
In nature, 5-ALA biosynthesis mainly has following two approach: i.e. C4 approach and C5 approach.
C4 approach is to be condensed generation 5-ALA under 5-Aminolevulinate synthase effect by succinyl CoA and glycine,
This approach is present in animal, fungus and non-sulfur photosynthetic bacteria.C5 approach is then under tRNA participates in, through three-step reaction
Catalysis generates 5-ALA, and this approach is widely present in plant, algae and antibacterial, including escherichia coli and paddy ammonia
Acid bar bacterium.
At present, only have and utilize C5 approach to produce the report of 5-ALA about Corynebacterium glutamicum on a small quantity, but
This approach is more complicated, and yield and the yield of 5-ALA are the lowest.
According to retrieval, the most not yet it is related to Corynebacterium glutamicum and utilizes C4 approach to produce the report of 5-ALA.
Summary of the invention
It is an object of the invention to overcome the deficiencies in the prior art, it is provided that a kind of glutamic acid rod producing 5-ALA
The construction method of bacillus strain.
Second object of the present invention is to provide a kind of Corynebacterium glutamicum strain producing 5-ALA.
Third object of the present invention is to provide answering of a kind of Corynebacterium glutamicum strain producing 5-ALA
With.
Technical scheme is summarized as follows:
The construction method of a kind of Corynebacterium glutamicum strain producing 5-ALA, comprises the steps:
(1) in Corynebacterium glutamicum (C.glutamicum) ATCC 13032, lactic dehydrogenase enzyme coding gene ldhA is knocked out
Generating gene pta-ackA, pqo and cat with acetic acid, the Strain Designation obtained is CB4；Phosphoenolpyruvate third in CB4 bacterial strain
Keto acid carboxylase encoding gene ppc is previously inserted into strong sod promoter, obtains bacterial strain CB5；Phosphoric acid alkene is knocked out in CB5 bacterial strain
Alcohol of formula pyruvate carboxykinase encoding gene pck, obtains bacterial strain CB6；
(2) the plasmid pXA and process LAN RhtA of the process LAN 5-Aminolevulinate synthase gene built are transported egg
White plasmid pEP2tuf-rhtA is transferred in CB6 bacterial strain, it is thus achieved that produce the Corynebacterium glutamicum strain of 5-ALA
AL2。
The Corynebacterium glutamicum strain AL2 producing 5-ALA that said method builds.
The use of the Corynebacterium glutamicum strain AL2 fermenting and producing 5-ALA of above-mentioned production 5-ALA
On the way.Beneficial effect
The present invention constructs a strain and utilizes C4 approach to produce the Corynebacterium glutamicum strain of 5-ALA, this bacterial strain
Can produce 2.78g/L 5-ALA in the culture medium with 10g/L glucose as carbon source, this is subsequent fermentation tank
Yield and the productivity of continuous feeding raising 5-ALA are laid a good foundation.
Accompanying drawing explanation
Fig. 1 is genetic manipulation target spot.
Fig. 2 A is the digestion verification collection of illustrative plates of plasmid pXA, and Fig. 2 B is plasmid pEP2tuf-rhtA digestion verification collection of illustrative plates.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention will be further described, and following embodiment is the technology in order to make this area
Personnel better understood when the present invention, but is not intended to be limited in any the present invention.
Used in the present invention:
Original strain Corynebacterium glutamicum (C.glutamicum) ATCC 13032 is purchased from ATCC (American Type
Culture Collection, http://www.atcc.org/)；
E.coli MG1655 be purchased from CGSC (Coli Genetic Stock Center, http: //
cgsc.biology.yale.edu/)。
Original plasmid pK18mobsacB, pEC-XK99E, pXMJ19 and pEP2 buy in BioVector NTCC company
(http://www.biovector.net/)。
5-ALA standard substance are from sigma company (http://www.sigmaaldrich.com/sigma-
Aldrich) buy.Restricted enzyme used, dephosphorylation enzyme, DNA ligase equimolecular biological reagent are public from Thermo
Department buys (http://www.thermoscientificbio.com/fermentas), and other biochemical reagents used are raw from raw work
Thing engineering (Shanghai) limited company buys (http://www.sangon.com/).
Embodiment 1: knock out the structure of plasmid pD-sacB
First the pK18mobsacB linear fragment after cutting using HindIII is as template, with following primer sacB-1 (SEQ
ID NO.1)/sacB-2 (SEQ ID NO.2) expands sacB gene.By sacB genetic fragment after MunI/EcoRV double digestion
It is attached with the plasmid pEC-XK99E after EcoRI/SmalI double digestion, obtains plasmid pEC-XK99E-sacB.With such as
Under primer trcsacB-1 (SEQ ID NO.3)/trcsacB-2 (SEQ ID NO.4), with pEC-XK99E-sacB plasmid make
The trcsacB fragment of trc promoter is contained for template amplification.
With following primer pD-1 (SEQ ID NO.5)/pD-2 (SEQ ID NO.6), using pK18mobsacB plasmid as
Template amplification contains the pD fragment of kalamycin resistance and escherichia coli replicon, finally by the fragment through AatII enzyme action
TrcsacB is attached with the pD fragment through identical enzyme action, obtains plasmid pD-sacB.
Embodiment 2: lactic dehydrogenase enzyme coding gene ldhA knock out with acetic acid constructive ways gene pta-ackA, pqo and
Cat knocks out
Lactic dehydrogenase enzyme coding gene ldhA knocks out:
With Corynebacterium glutamicum (C.glutamicum) ATCC 13032 genome as template, with ldh-1 (SEQ ID
NO.7)/ldh-2 (SEQ ID NO.8) is the fragment upstream of primer amplification gene ldhA, ldh-3 (SEQ ID NO.9)/ldh-4
(SEQ ID NO.10) is the segments downstream of primer amplification gene ldhA.After two fragments are cut glue recovery, with equimolar ratio
Fragment be template, with ldh-1/ldh-4 as primer, amplification obtains the fusion product of two fragments.Fragment after merging is used
EcoRI/HindIII double digestion with through as pD-sacB after double digestion connect, obtain plasmid pD-ldhA.
PD-ldhA plasmid is transferred in Corynebacterium glutamicum (C.glutamicum) ATCC 13032, uses kanamycin
The screening successful positive colony of restructuring, the transformant chosen is inoculated in 5mL BHIS fluid medium, and 30 DEG C, 220rpm is overnight
Cultivate, bacterium solution is diluted certain multiple and is coated on BHIS-Sucrose solid plate.By the bacterium colony that grows on flat board to point
The BHIS solid plate of nonreactive and the BHIS solid plate containing 25 μ g/mL kanamycin.Select on nonreactive flat board growth and
The colony inoculation that kanamycin flat board does not grows, in 5mL BHIS fluid medium, extracts genome, uses primer ldh-1/
Ldh-4 carries out PCR checking, obtains ldhA gene knock-out bacterial strain CB1.
Knocking out of pta-ackA operon
With C.glutamicum ATCC13032 genome as template, with ackA-1 (SEQ ID NO.11)/ackA-2
(SEQ ID NO.12) is the fragment upstream of primer amplification operon pta-ackA, ackA-3 (SEQ ID NO.13)/ackA-4
(SEQ ID NO.14) is the segments downstream of primer amplification operon pta-ackA.Two fragments are cut glue reclaim after, with etc. rub
Your fragment of ratio is template, and with ackA-1/ackA-4 as primer, amplification obtains the fusion product of two fragments.After merging
Fragment SalI/XbaI double digestion with through as pD-sacB plasmid after double digestion connect, obtain pta-ackA operon
Knock out plasmid pD-pta.
Being transferred in bacterial strain CB1 by pD-pta plasmid, with the kanamycin screening successful positive colony of restructuring, that chooses turns
Beggar is inoculated in 5mL BHIS fluid medium, 30 DEG C, 220rpm incubated overnight, bacterium solution dilutes certain multiple and is coated on
On BHIS-Sucrose solid plate.The bacterium colony grown on flat board to the BHIS solid plate of a nonreactive and is contained 25 μ g/mL
The BHIS solid plate of kanamycin.Select growth on nonreactive flat board and colony inoculation that kanamycin flat board does not grows arrives
In 5mL BHIS fluid medium, extract genome, use primer ackA-1/ackA-4 to carry out PCR checking, obtain pta-ackA
Operon knock-out bacterial strain CB2.
Knocking out of pqo gene
With C.glutamicum ATCC13032 genome as template, with pqo-1 (SEQ ID NO.15)/pqo-2 (SEQ
ID NO.16) it is the fragment upstream of primer amplification pqo gene, pqo-3 (SEQ ID NO.17)/pqo-4 (SEQ ID NO.18)
Segments downstream for primer amplification pqo.Two fragments are cut after glue reclaims, with the fragment of equimolar ratio as template, with pqo-
1/pqo-4 is primer, and amplification obtains the fusion product of two fragments.Fragment XbaI/PstI double digestion and warp after merging
Crossing the pD-sacB after same double digestion to connect, obtain pqo gene knocks out plasmid pD-pqo.
Being transferred in bacterial strain CB2 by pD-pqo plasmid, with the kanamycin screening successful positive colony of restructuring, that chooses turns
Beggar is inoculated in 5mL BHIS fluid medium, 30 DEG C, 220rpm incubated overnight, bacterium solution dilutes certain multiple and is coated on
On BHIS-Sucrose solid plate.The bacterium colony grown on flat board to the BHIS solid plate of a nonreactive and is contained 25 μ g/mL
The BHIS solid plate of kanamycin.Select growth on nonreactive flat board and colony inoculation that kanamycin flat board does not grows arrives
In 5mL BHIS fluid medium, extract genome, use primer pqo-1/pqo-4 to carry out PCR checking, obtain pqo gene
Knock-out bacterial strain CB3.
Knocking out of cat gene
With C.glutamicum ATCC13032 genome as template, with cat-1 (SEQ ID NO.19)/cat-2 (SEQ
ID NO.20) it is the fragment upstream of primer amplification cat gene, cat-3 (SEQ ID NO.21)/cat-4 (SEQ ID NO.22)
Segments downstream for primer amplification cat.Two fragments are cut after glue reclaims, with the fragment of equimolar ratio as template, with cat-
1/cat-4 is primer, and amplification obtains the fusion product of two fragments.Fragment XbaI/SalI double digestion and warp after merging
Crossing the pD-sacB after same double digestion to connect, obtain cat gene knocks out plasmid pD-cat.
Being transferred in bacterial strain CB3 by pD-cat plasmid, with the kanamycin screening successful positive colony of restructuring, that chooses turns
Beggar is inoculated in 5mL BHIS fluid medium, 30 DEG C, 220rpm incubated overnight, bacterium solution dilutes certain multiple and is coated on
On BHIS-Sucrose solid plate.The bacterium colony grown on flat board to the BHIS solid plate of a nonreactive and is contained 25 μ g/mL
The BHIS solid plate of kanamycin.Select growth on nonreactive flat board and colony inoculation that kanamycin flat board does not grows arrives
In 5mL BHIS fluid medium, extract genome, use primer cat-1/cat-4 to carry out PCR checking, obtain cat gene
Knock-out bacterial strain CB4.
Wherein BHIS medium component is (g/L): Medulla Bovis seu Bubali heart leaching powder 37, sorbitol 91.
BHIS solid culture based component is (g/L): Medulla Bovis seu Bubali heart leaching powder 37, sorbitol 91, agar 2% (W/V)
BHIS-Sucrose solid medium (g/L): Medulla Bovis seu Bubali heart leaching powder 37, sorbitol 91, agar 2% (W/V), sugarcane
Sugar 10% (W/V).
Embodiment 3: be previously inserted into strong sod promoter and PCK coding base at ppc gene
Because of knocking out of pck gene
Ppc gene is previously inserted into strong sod promoter
With C.glutamicum ATCC13032 genome as template, with ppc-1 (SEQ ID NO.23)/ppc-2 (SEQ
ID NO.24) it is the fragment upstream of primer amplification gene ppc.sod-1(SEQ ID NO.25)/sod-2(SEQ ID NO.26)
For expanding the promoter of sod gene.Ppc-3 (SEQ ID NO.27)/ppc-4 (SEQ ID NO.28) is used for expanding ppc base
3 fragments are cut after glue reclaims, with the fragment of equimolar ratio as template, with ppc-1/ppc-4 are by the segments downstream of cause respectively
Primer, amplification obtains the fusion product of three fragments.Fragment after merging is with same with process after XbaI/HindIII double digestion
Plasmid vector pD-sacB after sample double digestion connects.Obtain plasmid pD-ppc.
Electricity is utilized to be transferred in bacterial strain CB4 the plasmid built, with the most positive gram of kanamycin screening restructuring
Grand, the transformant chosen is inoculated in 5mL BHIS fluid medium, 30 DEG C, 220rpm incubated overnight, by certain for bacterium solution dilution
Multiple be coated on BHIS-Sucrose solid plate.By the bacterium colony that grows on flat board to the BHIS solid plate of a nonreactive and
BHIS solid plate containing 25 μ g/mL kanamycin.The bacterium that kanamycin flat board does not grows by growth on nonreactive flat board
Fall to being inoculated in 5mL BHIS fluid medium, extract genome, send order-checking, obtain being previously inserted into sod promoter at ppc gene
Bacterial strain CB5.
Knocking out of pck gene
With C.glutamicum ATCC 13032 genome as template, carry out PCR amplification with following primer.pck-1(SEQ
ID NO.29)/pck-2 (SEQ ID NO.30) is for the fragment upstream of amplification gene pck.pck-3(SEQ ID NO.31)/
Pck-4 (SEQ ID NO.32) is for the segments downstream of amplification gene pck.By fragment upstream after EcoRI/XbaI double digestion
It is connected with the pD-sacB plasmid after identical double digestion, obtains plasmid pD-pck (F), plasmid pD-pck (F) warp that will build
It is attached with the segments downstream through PstI/HindIII double digestion after crossing PstI/HindIII double digestion, obtains pD-pck matter
Grain.
This plasmid is transferred in bacterial strain CB5, by the kanamycin screening successful positive colony of restructuring, the transformant chosen
It is inoculated in 5mL BHIS fluid medium, 30 DEG C, 220rpm incubated overnight, bacterium solution is diluted certain multiple and is coated on
On BHIS-Sucrose solid plate.The bacterium colony grown on flat board to the BHIS solid plate of a nonreactive and is contained 25 μ g/mL
The BHIS solid plate of kanamycin.The colony inoculation that kanamycin flat board does not grows by growth on nonreactive flat board arrives
In 5mLBHIS fluid medium, extract genome, use primer pck-1/pck-4 to carry out PCR checking, obtainpckStriking of gene
Degerming strain CB6.
Embodiment 4: plasmid pXA and pEP2tufThe structure of-rhtA
The structure of pXA plasmid
By the 5-Aminolevulinate synthase gene hemA of hydrogenlike silicon ion (Rhodobacter sphaeroides) according to
The codon preference of Corynebacterium glutamicum carries out codon optimized, and the method for the gene full genome synthesis after optimizing is carried out
Synthesis (as shown in SEQ ID NO.39), utilizes primer hemA-1 (SEQ ID NO.33)/hemA-2 (SEQ ID NO.34) to expand
Increase the hemA fragment after optimizing, by the hemA fragment that obtains after PstI/XbaI double digestion with after identical double digestion
Shuttle plasmid pXMJ19 connects, and obtains pXA plasmid, and Fig. 2 A is the digestion verification collection of illustrative plates of plasmid pXA.
With C.glutamicum ATCC 13032 genome as template, with tuf-1 (SEQ ID NO.35)/tuf-2 (SEQ
ID NO.36) be primer amplification tuf promoter, by after tuf fragment EcoRI/SalI enzyme action with the pEP2 through identical enzyme action
Plasmid connects, and obtains the low copy plasmid pEP2 with tuf promotertuf.With E.coli MG1655 genome as template, use
RhtA-1 (SEQ ID NO.37)/rhtA-2 (SEQ ID NO.38) is primer amplification rhtA fragment, by fragment XbaI/
With the pEP2 through identical enzyme action after BamHI double digestiontufPlasmid is attached, and obtains pEP2tuf-rhtA plasmid, Fig. 2 B is matter
Grain pEP2tuf-rhtA digestion verification collection of illustrative plates.
Import to, in bacterial strain CB6, utilize chloromycetin to screen by the pXA plasmid built, obtain with pXA plasmid
Strains A L1.The pEP2 that will buildtuf-rhtA plasmid utilizes electricity to turn and imports in AL1, utilize chloromycetin and kanamycin to carry out
Screening, obtains final 5-ALA and produces strains A L2.
Fig. 1 is shown in the genetic manipulation that the present invention relates to, and in Fig. 1, " X " expression knocks out, and underscore represents process LAN.
Table 1 strain construction the primer sequence
Embodiment 5: utilize the bacterial strain built to carry out the shake flask fermentation of 5-ALA
Strains A L2 built is fermented in shaking flask.
Concrete fermentation mode is as follows
Vaccination ways is: first activated strains on BHIS solid plate, 30 DEG C cultivate to bacterium colony can apparent time, choose single bacterium
Fall the BHIS liquid of the chloromycetin being seeded to the kanamycin sulfate equipped with the final concentration of 25 μ g/mL of 5mL and final concentration of 10 μ g/mL
After body culture medium cultivates 15h, it is transferred in M1 culture medium, when cultivating to logarithm middle and late stage, seed is inoculated into M2 and cultivates
In base.Culture medium is added the isopropylthio-β-D-galactoside of final concentration of 0.5mM as derivant.After fermentation ends
It is 2.78g/L that detection generates the yield of 5-ALA.
M1 medium component is (g/L): glucose 10, yeast extract 10, tryptone 10, NaCl 2.5.
M2 medium component is (g/L): glucose 10, yeast extract 10, tryptone 10, NaCl 2.5,3-(N-
Quinoline) propane sulfonic acid 21, glycine 7.5.
Claims (3)
1. produce a construction method for the Corynebacterium glutamicum strain of 5-ALA, it is characterized in that including walking as follows
Rapid:
(1) in Corynebacterium glutamicum (C.glutamicum) ATCC 13032, lactic dehydrogenase enzyme coding gene ldhA and second are knocked out
Acid generates gene pta-ackA, pqo and cat, and the Strain Designation obtained is CB4；Phosphoenolpyruvic acid in CB4 bacterial strain
Carboxylase encoding gene ppc is previously inserted into strong sod promoter, obtains bacterial strain CB5；Phosphoenolpyruvate is knocked out in CB5 bacterial strain
Pyruvate carboxykinase encoding gene pck, obtains bacterial strain CB6；
(2) by the plasmid pXA of process LAN 5-Aminolevulinate synthase gene built and process LAN RhtA transport protein
Plasmid pEP2tuf-rhtA is transferred in CB6 bacterial strain, it is thus achieved that produce the Corynebacterium glutamicum strain AL2 of 5-ALA.
2. the Corynebacterium glutamicum strain AL2 producing 5-ALA that the method described in claim 1 builds.
3. the purposes of the Corynebacterium glutamicum strain AL2 producing 5-ALA of claim 2.
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Cited By (7)
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| CN106434514A (en) * | 2016-11-09 | 2017-02-22 | 天津大学 | Corynebacterium glutamicum engineering strain for producing 5-aminolevulinic acid |
| CN106434513A (en) * | 2016-11-09 | 2017-02-22 | 天津大学 | Corynebacterium glutamicum recombinant strain for producing 5-aminolevulinic acid |
| CN107012161A (en) * | 2017-04-03 | 2017-08-04 | 天津大学 | Corynebacterium glutamicum and structure and application using stalk hydrolyzate high yield butanedioic acid |
| CN108517327A (en) * | 2018-04-20 | 2018-09-11 | 中国科学院天津工业生物技术研究所 | 5-ALA superior strain and its preparation method and application |
| WO2020186326A1 (en) * | 2019-03-21 | 2020-09-24 | De Leao Rosenmann Bernardo | Cosmetic formulations formed by a nutritive mixture from a fermentative process |
| WO2020232519A1 (en) * | 2019-05-22 | 2020-11-26 | De Leao Rosenmann Bernardo | Nutritional compound formed by bacterial fermentation content for use as a supplement or additive for animal feed |
| CN115044602A (en) * | 2022-05-23 | 2022-09-13 | 天津大学 | Strain for anaerobic synthesis of corynebacterium 5-aminolevulinic acid glutamate and construction method |
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| CN106434513A (en) * | 2016-11-09 | 2017-02-22 | 天津大学 | Corynebacterium glutamicum recombinant strain for producing 5-aminolevulinic acid |
| CN107012161A (en) * | 2017-04-03 | 2017-08-04 | 天津大学 | Corynebacterium glutamicum and structure and application using stalk hydrolyzate high yield butanedioic acid |
| CN108517327A (en) * | 2018-04-20 | 2018-09-11 | 中国科学院天津工业生物技术研究所 | 5-ALA superior strain and its preparation method and application |
| WO2020186326A1 (en) * | 2019-03-21 | 2020-09-24 | De Leao Rosenmann Bernardo | Cosmetic formulations formed by a nutritive mixture from a fermentative process |
| WO2020232519A1 (en) * | 2019-05-22 | 2020-11-26 | De Leao Rosenmann Bernardo | Nutritional compound formed by bacterial fermentation content for use as a supplement or additive for animal feed |
| CN115044602A (en) * | 2022-05-23 | 2022-09-13 | 天津大学 | Strain for anaerobic synthesis of corynebacterium 5-aminolevulinic acid glutamate and construction method |
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