CN106636171A - Crynebacterium glutamicum engineering strain for producing 5-aminolevulinic acid and construction method thereof+ - Google Patents
Crynebacterium glutamicum engineering strain for producing 5-aminolevulinic acid and construction method thereof+ Download PDFInfo
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Abstract
The invention discloses a crynebacterium glutamicum engineering strain for producing 5-aminolevulinic acid and a construction method thereof. The construction method comprises the steps of 1, knocking out lactic dehydrogenase encoding gene idhA, and acetic acid generation genes pta-ackA, pqo and cat in the corynebacterium glutamicum, inserting a strong sod promoter in front of a phosphoenolpyruvate carboxylase encoding gene ppc, knocking out a phosphoenolpyruvate carboxykinase pck, thus acquiring the strain CB6; 2, knocking out a penicillin-binding protein encoding gene pbpla in the strain CB6, thus acquiring the strain CB7; transferring the plasmid pXA of a built over-expressed 5-aminolevulinic acid synthase gene into the strain CB7, thus acquiring the engineering strain L2. According to the engineering strain provided by the invention, the 5-aminolevulinic acid is produced in a medium in which 10g/L glucose is added as a carbon source; compared with reference strains, the engineering strain is respectively improved by 13.53% and above.
Description
Technical field
The invention belongs to biotechnology and application, more particularly to a kind of paddy ammonia for producing 5-ALA
Sour bar bacterium engineered strain and structure and application.
Background technology
5-ALA, molecular weight is 131.13, and fusing point is 118 DEG C.It is a kind of non-protein amino acid.Due to 5- ammonia
Base levulic acid have Small side effects, good penetrability the characteristics of, be widely used in cutaneum carcinoma, carcinoma of urinary bladder, digestion
In road cancer, the diagnosis of lung cancer and optical dynamic therapy (PDT).
5-ALA is the precursor of biological azole compounds in vivo, is had a wide range of applications.Agriculturally, by
Easily degrade in the environment in 5-ALA, it is harmless to mammal, can selectively kill off the insect pests, therefore can make
It is widely used for photodynamics insecticide.In addition, 5-ALA is improving the freeze proof evil of crops and resistance to
The aspect such as salt ability and coordinate plant growth also receives the extensive concern of people.In medical domain, 5-ALA
As second generation sensitising agent, the treatment of cutaneum carcinoma locally or systemically is can be not only used for, it may also be used for carcinoma of urinary bladder, digestive system cancer
And the diagnosis of the cancer such as lung cancer.
PBP (Penicillin binding proteins, PBPs) is present on bacterial cytoplasm film
A class small proteinaceous.The albuminoid can be named with the covalently bound property of penicillin according to it.PBPs is
The end reaction enzyme of peptide glycan synthesis in cell membrane, its presence maintains normal form and function to play to pass bacterial cell
Important effect.
Corynebacterium glutamicum includes at least 9 PBPs, is divided into HMW PBPs (HMW-PBPs) and low-molecular-weight PBPs
(LMW-PBPs), wherein HMW-PBPs is divided into A classes HMW-PBPs and B classes HMW-PBPs again, A classes HMW-PBPs comprising PBP1a and
PBP1b, with transpeptidase and transglycosyiase activity.B classes HMW-PBPs have transpeptidase functional domain and a unknown function domain, bag
Include PBP2a, pbp2b and FtsI.It is generally acknowledged that A classes HMW-PBPs are responsible for the extension of cell, and B classes HMW-PBPs are responsible for cell point
The formation of change process septation.
According to retrieval, not yet there is the report that 5-ALA is produced using PBP knock-out bacterial strain now.
The content of the invention
The purpose of the present invention is to overcome the deficiencies in the prior art, there is provided a kind of glutamic acid rod for producing 5-ALA
The construction method of bacillus engineered strain.
Second object of the present invention is to provide a kind of Corynebacterium glutamicum engineered strain for producing 5-ALA.
Third object of the present invention is to provide a kind of Corynebacterium glutamicum engineered strain for producing 5-ALA
Application.
Technical scheme is summarized as follows：
The construction method of the Corynebacterium glutamicum engineered strain of production 5-ALA, comprises the steps：
(1) lactic dehydrogenase enzyme coding gene ldhA is knocked out in Corynebacterium glutamicum (C.glutamicum) ATCC 13032
Gene pta-ackA, pqo and cat are generated with acetic acid, the Strain Designation for obtaining is CB4；Phosphoenolpyruvate third in CB4 bacterial strains
Keto acid carboxylase encoding gene ppc is previously inserted into strong sod promoters, obtains bacterial strain CB5；Phosphoric acid alkene is knocked out in CB5 bacterial strains
Alcohol of formula pyruvate carboxykinase encoding gene pck, obtains bacterial strain CB6；
(2) PBP encoding gene pbp1a is knocked out in bacterial strain CB6, bacterial strain CB7 is obtained；In bacterial strain CB7
The plasmid pXA of the overexpression 5-Aminolevulinate synthase gene for building is proceeded to, obtains producing the paddy of 5-ALA
Propylhomoserin bar bacterium engineered strain L2；
Or PBP encoding gene pbp1b is knocked out in bacterial strain CB6, obtain bacterial strain CB8；In bacterial strain CB8
The plasmid pXA of the overexpression 5-Aminolevulinate synthase gene for building is proceeded to, obtains producing the paddy of 5-ALA
Propylhomoserin bar bacterium engineered strain L3；
Or PBP encoding gene pbp2b is knocked out in bacterial strain CB6, obtain bacterial strain CB9；In bacterial strain CB9
The plasmid pXA of the overexpression 5-Aminolevulinate synthase gene for building is proceeded to, obtains producing the paddy of 5-ALA
Propylhomoserin bar bacterium engineered strain L4.
The Corynebacterium glutamicum engineered strain of the production 5-ALA that said method builds.
The Corynebacterium glutamicum engineered strain fermenting and producing 5-ALA of above-mentioned production 5-ALA
Purposes.
The engineered strain of the present invention has knocked out PBP encoding gene, is carbon in the glucose of addition 10g/L
In the culture medium in source, engineered strain L2, L3 and L4 of structure are able to produce 2.35g/L, 2.68g/L and 2.53g/L 5- ammonia
Base levulic acid, than control strain 13.53%, 29.47% and 22.22% has been respectively increased.
Description of the drawings
Fig. 1 is the digestion verification collection of illustrative plates of plasmid pXA.
Fig. 2A is the PCR checking collection of illustrative plates that gene pbp1a is knocked out, and 2B is the PCR checking collection of illustrative plates that gene pbp1b is knocked out,
2C is the PCR checking collection of illustrative plates that gene pbp2b is knocked out.
Fig. 3 is the fermentation results of recombinant bacterial strain L1, L2, L3 and L4 in shaking flask.
Specific embodiment
With reference to specific embodiment, the present invention will be further described, and following embodiments are the technologies in order that this area
Personnel better understood when the present invention, but the present invention is not intended to be limited in any.
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 are purchased from CGSC (Coli Genetic Stock Center, http://
cgsc.biology.yale.edu/)。
Original plasmid pK18mobsacB, pEC-XK99E, pXMJ19 and pEP2 are bought in BioVector NTCC companies
(http://www.biovector.net/)。
5-ALA standard items are from sigma companies (http://www.sigmaaldrich.com/sigma-
Aldrich) buy.
Restriction enzyme used, dephosphorylation enzyme, DNA ligase equimolecular biological reagent are bought from Thermo companies
(http://www.thermoscientificbio.com/fermentas), other biochemical reagents used are from raw work bioengineering
(Shanghai) limited company buys (http://www.sangon.com/).
Embodiment 1：Knock out the structure and overexpression 5-Aminolevulinate synthase gene plasmid pXA of plasmid pD-sacB
Build
The structure of pD-sacB plasmids
First using the pK18mobsacB linear fragments after HindIII cuttings as template, with following primer sacB-1 (SEQ
ID NO.1)/sacB-2 (SEQ ID NO.2) amplification sacB genes.By sacB genetic fragments after MunI/EcoRV double digestions
It is attached with the plasmid pEC-XK99E after EcoRI/SmalI double digestions, 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 plasmids work
For the trcsacB fragments that template amplification contains trc promoters.
With following primer pD-1 (SEQ ID NO.5)/pD-2 (SEQ ID NO.6), using pK18mobsacB plasmids as
Template amplification contains the pD fragments of kalamycin resistance and Escherichia coli replicon, finally by through the fragment of AatII digestions
TrcsacB is attached with the pD fragments through identical digestion, obtains plasmid pD-sacB.
The structure of pXA plasmids
By the 5-Aminolevulinate synthase gene hemA of hydrogenlike silicon ion (Rhodobacter sphaeroides) according to
The codon preference of Corynebacterium glutamicum carries out codon optimization, and the method that the gene after optimization is synthesized with full genome is carried out
Synthesis is expanded (as shown in SEQ ID NO.47) using primer hemA-1 (SEQ ID NO.7)/hemA-2 (SEQ ID NO.8)
HemA fragments after optimization, by the hemA fragments for obtaining after PstI/XbaI double digestions with wearing after identical double digestion
Shuttle plasmid pXMJ19 connects, and obtains pXA plasmids, and the digestion verification collection of illustrative plates of plasmid pXA is shown in Fig. 1.
Embodiment 2：The knockout and acetic acid constructive ways gene pta-ackA, pqo of lactic dehydrogenase enzyme coding gene ldhA and
The knockout of cat
The knockout of lactic dehydrogenase enzyme coding gene ldhA：
With the genomes of Corynebacterium glutamicum (C.glutamicum) ATCC 13032 as template, with ldh-1 (SEQ ID
NO.9)/ldh-2 (SEQ ID NO.10) for primer amplification gene ldhA fragment upstream, ldh-3 (SEQ ID NO.11)/
Ldh-4 (SEQ ID NO.12) is the segments downstream of primer amplification gene ldhA.Two fragments are cut after glue reclaim, with equimolar
The fragment of ratio is template, and with ldh-1/ldh-4 as primer, amplification obtains the fusion product of two fragments.By the piece after fusion
Section is connected with EcoRI/HindIII double digestions with the pD-sacB after same double digestion, obtains plasmid pD-ldhA.
PD-ldhA plasmids are transferred in C.glutamicum ATCC 13032, it is successful with kanamycins screening restructuring
Positive colony, the transformant chosen is inoculated in 5mL BHIS fluid nutrient mediums, 30 DEG C, and 220rpm incubated overnights are dilute by bacterium solution
Release certain multiple to be coated on BHIS-Sucrose solid plates.By BHIS solid of the bacterium colony grown on flat board to a nonreactive
Flat board and the BHIS solid plates containing 25 μ g/mL kanamycins.Select on nonreactive flat board growth and kanamycins flat board not
The colony inoculation of growth extracts genome in 5mL BHIS fluid nutrient mediums, enters performing PCR using primer ldh-1/ldh-4 and tests
Card, obtains ldhA gene knock-out bacterial strain CB1.
The knockout of pta-ackA operators
With C.glutamicum ATCC13032 genomes as template, with ackA-1 (SEQ ID NO.13)/ackA-2
(SEQ ID NO.14) expands the fragment upstream of operator pta-ackA, ackA-3 (SEQ ID NO.15)/ackA-4 for primer
(SEQ ID NO.16) is the segments downstream that primer expands operator pta-ackA.Two fragments are cut after glue reclaim, with etc. rub
The fragment of your ratio is template, and with ackA-1/ackA-4 as primer, amplification obtains the fusion product of two fragments.After merging
Fragment be connected with the pD-sacB plasmids after same double digestion with SalI/XbaI double digestions, obtain pta-ackA operators
Knock out plasmid pD-pta.
PD-pta plasmids are transferred in bacterial strain CB1, with the successful positive colony of kanamycins screening restructuring, that what is chosen turns
Beggar is inoculated in 5mL BHIS fluid nutrient mediums, 30 DEG C, 220rpm incubated overnights, bacterium solution is diluted into certain multiple and is coated on
On BHIS-Sucrose solid plates.By the bacterium colony grown on flat board to the BHIS solid plates of a nonreactive and containing 25 μ g/mL
The BHIS solid plates of kanamycins.Select the growth on nonreactive flat board and colony inoculation that kanamycins flat board does not grow is arrived
In 5mL BHIS fluid nutrient mediums, genome is extracted, using primer ackA-1/ackA-4 performing PCR checking is entered, obtain pta-ackA
Operator knock-out bacterial strain CB2.
The knockout of pqo genes
With C.glutamicum ATCC13032 genomes as template, with pqo-1 (SEQ ID NO.17)/pqo-2 (SEQ
ID NO.18) for primer expand pqo genes fragment upstream, pqo-3 (SEQ ID NO.19)/pqo-4 (SEQ ID NO.20)
For the segments downstream that primer expands pqo.Two fragments are cut after glue reclaim, the fragment with equimolar ratio as template, with pqo-
1/pqo-4 is primer, and amplification obtains the fusion product of two fragments.By the fragment after fusion XbaI/PstI double digestions and Jing
The pD-sacB connections crossed after same double digestion, obtain the knockout plasmid pD-pqo of pqo genes.
PD-pqo plasmids are transferred in bacterial strain CB2, with the successful positive colony of kanamycins screening restructuring, that what is chosen turns
Beggar is inoculated in 5mL BHIS fluid nutrient mediums, 30 DEG C, 220rpm incubated overnights, bacterium solution is diluted into certain multiple and is coated on
On BHIS-Sucrose solid plates.By the bacterium colony grown on flat board to the BHIS solid plates of a nonreactive and containing 25 μ g/mL
The BHIS solid plates of kanamycins.Select the growth on nonreactive flat board and colony inoculation that kanamycins flat board does not grow is arrived
In 5mL BHIS fluid nutrient mediums, genome is extracted, using primer pqo-1/pqo-4 performing PCR checking is entered, obtain pqo genes
Knock-out bacterial strain CB3.
The knockout of cat genes
With C.glutamicum ATCC13032 genomes as template, with cat-1 (SEQ ID NO.21)/cat-2 (SEQ
ID NO.22) for primer expand cat genes fragment upstream, cat-3 (SEQ ID NO.23)/cat-4 (SEQ ID NO.24)
For the segments downstream that primer expands cat.Two fragments are cut after glue reclaim, the fragment with equimolar ratio as template, with cat-
1/cat-4 is primer, and amplification obtains the fusion product of two fragments.By the fragment after fusion XbaI/SalI double digestions and Jing
The pD-sacB connections crossed after same double digestion, obtain the knockout plasmid pD-cat of cat genes.
PD-cat plasmids are transferred in bacterial strain CB3, with the successful positive colony of kanamycins screening restructuring, that what is chosen turns
Beggar is inoculated in 5mL BHIS fluid nutrient mediums, 30 DEG C, 220rpm incubated overnights, bacterium solution is diluted into certain multiple and is coated on
On BHIS-Sucrose solid plates.By the bacterium colony grown on flat board to the BHIS solid plates of a nonreactive and containing 25 μ g/mL
The BHIS solid plates of kanamycins.Select the growth on nonreactive flat board and colony inoculation that kanamycins flat board does not grow is arrived
In 5mL BHIS fluid nutrient mediums, genome is extracted, using primer cat-1/cat-4 performing PCR checking is entered, obtain cat genes
Knock-out bacterial strain CB4.
Wherein BHIS medium components are (g/L)：The bovine brain heart soaks powder 37, sorbierite 91.
BHIS solid cultures based component is (g/L)：The bovine brain heart soaks powder 37, sorbierite 91, agar 2% (W/V)
BHIS-Sucrose solid mediums (g/L)：The bovine brain heart soaks powder 37, sorbierite 91, agar 2% (W/V), sugarcane
10% (W/V) of sugar.
Embodiment 3：Strong sod promoters and PCK coding base are previously inserted in ppc genes
Because of the knockout of pck
Ppc genes are previously inserted into strong sod promoters
With C.glutamicum ATCC13032 genomes as template, with ppc-1 (SEQ ID NO.25)/ppc-2 (SEQ
ID NO.26) for primer amplification gene ppc fragment upstream.sod-1(SEQ ID NO.27)/sod-2(SEQ ID NO.28)
For expanding the promoter of sod genes.Ppc-3 (SEQ ID NO.29)/ppc-4 (SEQ ID NO.30) is used to expand ppc bases
The segments downstream of cause, respectively cuts 3 fragments after glue reclaim, and the fragment with equimolar ratio is with ppc-1/ppc-4 as template
Primer, amplification obtains the fusion product of three fragments.By the fragment after fusion with after XbaI/HindIII double digestions with through same
Plasmid vector pD-sacB connections after sample double digestion.Obtain plasmid pD-ppc.
The plasmid for building is transferred in bacterial strain CB4 using electricity, with successfully positive gram of kanamycins screening restructuring
Grand, the transformant chosen is inoculated in 5mL BHIS fluid nutrient mediums, 30 DEG C, 220rpm incubated overnights, and bacterium solution dilution is certain
Multiple be coated on BHIS-Sucrose solid plates.By the bacterium colony grown on flat board to the BHIS solid plates of a nonreactive and
BHIS solid plates containing 25 μ g/mL kanamycins.The bacterium that kanamycins flat board does not grow by the growth on nonreactive flat board
In falling to being inoculated into 5mL BHIS fluid nutrient mediums, genome is extracted, send sequencing, obtain being previously inserted into sod promoters in ppc genes
Bacterial strain CB5.
The knockout of pck genes
With the genomes of C.glutamicum ATCC 13032 as template, with following primer performing PCR amplification is entered.pck-1(SEQ
ID NO.31)/pck-2 (SEQ ID NO.32) be used for amplification gene pck fragment upstream.pck-3(SEQ ID NO.33)/
Pck-4 (SEQ ID NO.34) is used for the segments downstream of amplification gene pck.By fragment upstream after EcoRI/XbaI double digestions
Connect with the pD-sacB plasmids after identical double digestion, obtain plasmid pD-pck (F), plasmid pD-pck (F) Jing that will be built
Cross after PstI/HindIII double digestions and be attached with the segments downstream through PstI/HindIII double digestions, obtain pD-pck matter
Grain.
The plasmid is transferred in bacterial strain CB5, with the successful positive colony of kanamycins screening restructuring, the transformant chosen
In being inoculated in 5mL BHIS fluid nutrient mediums, 30 DEG C, bacterium solution is diluted certain multiple and is coated on by 220rpm incubated overnights
On BHIS-Sucrose solid plates.By the bacterium colony grown on flat board to the BHIS solid plates of a nonreactive and containing 25 μ g/mL
The BHIS solid plates of kanamycins.Will on nonreactive flat board growth and colony inoculation that kanamycins flat board does not grow to 5mL
In BHIS fluid nutrient mediums, genome is extracted, using primer pck-1/pck-4 performing PCR checking is entered, obtainedpckThe knockout of gene
Bacterial strain CB6.
PXA plasmids are transferred in CB6, are screened with chloramphenicol, obtain the 5-ALA with pXA plasmids
Production bacterial strain L1.
Embodiment 4：The knockout of pbp1a
With the genomes of C.glutamicum ATCC 13032 as template, with following primer performing PCR amplification is entered.pbp1a-1
(SEQ ID NO.35)/pbp1a-2 (SEQ ID NO.36) is used for the fragment upstream of amplification gene pbp1a, pbp1a-3 (SEQ
ID NO.37)/pbp1a-4 (SEQ ID NO.38) be used for amplification gene pbp1a segments downstream.With pbp1a-1/pbp1a-4
For primer, pbp1a fragment upstreams and segments downstream carry out fusion DNA vaccine for template.Fragment will be merged double through XbaI/HindIII
Connect with the pD-sacB plasmids after identical digestion after digestion.Obtain pbp1a and knock out plasmid pD-pbp1a.
The plasmid is transferred in bacterial strain CB6, with the successful positive colony of kanamycins screening restructuring, the transformant chosen
In being inoculated in 5mL BHIS fluid nutrient mediums, 30 DEG C, bacterium solution is diluted certain multiple and is coated on by 220rpm incubated overnights
On BHIS-Sucrose solid plates.By the bacterium colony grown on flat board to the BHIS solid plates of a nonreactive and containing 25 μ g/mL
The BHIS solid plates of kanamycins.Will on nonreactive flat board growth and colony inoculation that kanamycins flat board does not grow to 5mL
In BHIS fluid nutrient mediums, genome is extracted, using primer pbp1a-1/pbp1a-4 performing PCR checking is entered, obtain pbp1a genes
Knock-out bacterial strain CB7, see Fig. 2A.
PXA plasmids are transferred in CB7, are screened with chloramphenicol, obtain the 5-ALA with pXA plasmids
Production bacterial strain L2.
Embodiment 5：The knockout of pbp1b
With the genomes of C.glutamicum ATCC 13032 as template, with following primer performing PCR amplification is entered.pbp1b-1
(SEQ ID NO.39)/pbp1b-2 (SEQ ID NO.40) is used to expand the fragment upstream of pbp1b, pbp1b-3 (SEQ ID
NO.41)/pbp1b-4 (SEQ ID NO.42) is used for the segments downstream of amplification gene pbp1b.With pbp1b-1/pbp1b-4 to draw
Thing, pbp1b fragment upstreams and segments downstream carry out fusion DNA vaccine for template.By fusion fragment after BamHI/pstI double digestions
Connect with the pD-sacB plasmids after identical digestion, obtain gene pbp1b and knock out plasmid pD-pbp1b.
The plasmid is transferred in bacterial strain CB6, with the successful positive colony of kanamycins screening restructuring, the transformant chosen
In being inoculated in 5mL BHIS fluid nutrient mediums, 30 DEG C, bacterium solution is diluted certain multiple and is coated on by 220rpm incubated overnights
On BHIS-Sucrose solid plates.By the bacterium colony grown on flat board to the BHIS solid plates of a nonreactive and containing 25 μ g/mL
The BHIS solid plates of kanamycins.Will on nonreactive flat board growth and colony inoculation that kanamycins flat board does not grow to 5mL
In BHIS fluid nutrient mediums, genome is extracted, using primer pbp1b-1/pbp1b-4 performing PCR checking is entered, obtain pbp1b genes
Knock-out bacterial strain CB8, see Fig. 2 B.
PXA plasmids are transferred in CB8, are screened with chloramphenicol, obtain the 5-ALA with pXA plasmids
Production bacterial strain L3.
Embodiment 6：The knockout of pbp2b
With the genomes of C.glutamicum ATCC 13032 as template, with following primer performing PCR amplification is entered.pbp2b-1
(SEQ ID NO.43)/pbp2b-2 (SEQ ID NO.44) is used for the fragment upstream of amplification gene pbp2b, pbp2b-3 (SEQ
ID NO.45)/pbp2b-4 (SEQ ID NO.46) be used for amplification gene pbp2b segments downstream.With pbp2b-1/pbp2b-4
For primer, pbp2b fragment upstreams and segments downstream carry out fusion DNA vaccine for template.Fragment will be merged double through EcoRI/HindII
Connect with the pD-sacB after identical digestion after digestion, obtain gene pbp2b and knock out plasmid pD-pbp2b.
The plasmid is transferred in bacterial strain CB6, with the successful positive colony of kanamycins screening restructuring, the transformant chosen
In being inoculated in 5mL BHIS fluid nutrient mediums, 30 DEG C, bacterium solution is diluted certain multiple and is coated on by 220rpm incubated overnights
On BHIS-Sucrose solid plates.By the bacterium colony grown on flat board to the BHIS solid plates of a nonreactive and containing 25 μ g/mL
The BHIS solid plates of kanamycins.Will on nonreactive flat board growth and colony inoculation that kanamycins flat board does not grow to 5mL
In BHIS fluid nutrient mediums, genome is extracted, using primer pbp2b-1/pbp2b-4 performing PCR checking is entered, obtain pbp2b genes
Knock-out bacterial strain CB9, see Fig. 2 C.
PXA plasmids are transferred in CB9, are screened with chloramphenicol, obtain the 5-ALA with pXA plasmids
Production bacterial strain L4.
The strain construction the primer sequence of table 1
Embodiment 7：The shake flask fermentation of 5-ALA is carried out using the bacterial strain for building
By bacterial strain L1 (control), L2, L3, L4, (220rpm, 30 DEG C) is fermented in M2 culture mediums.
(L1：The plasmid pXA of overexpression 5-Aminolevulinate synthase gene hemA is proceeded in bacterial strain CB6,5- ammonia is obtained
Base levulic acid produces bacterial strain L1；)
Vaccination ways are：The activated strains first on BHIS solid plates, cultivate at 30 DEG C to bacterium colony can apparent time, choose single bacterium
Fall to being seeded in the test tube of the BHIS fluid nutrient mediums equipped with final concentration of 10 μ g/mL chloramphenicol and cultivate 15 hours or so, switching
To in M1 culture mediums, in culture to OD600When=10 or so, seed is inoculated into into M2 culture mediums and is fermented, added in culture medium
The chloramphenicol of final concentration of 10 μ g/mL and the isopropylthiogalactoside (IPTG) of 0.5mM.As a result Fig. 3 is seen.
M1 medium components are (g/L)：Glucose 10, yeast extract 10, tryptone 10, NaCl 2.5.
M2 medium components are (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. the construction method of the Corynebacterium glutamicum engineered strain of 5-ALA is produced, it is characterized in that including following step
Suddenly：
(1) lactic dehydrogenase enzyme coding gene ldhA and second are knocked out in Corynebacterium glutamicum (C.glutamicum) ATCC 13032
Acid generates gene pta-ackA, pqo and cat, and the Strain Designation for obtaining is CB4；PEP in CB4 bacterial strains
Carboxylation enzyme coding gene ppc is previously inserted into strong sod promoters, obtains bacterial strain CB5；Phosphoenolpyruvate is knocked out in CB5 bacterial strains
Pyruvate carboxykinase encoding gene pck, obtains bacterial strain CB6；
(2) PBP encoding gene pbp1a is knocked out in bacterial strain CB6, bacterial strain CB7 is obtained；Proceed in bacterial strain CB7
The plasmid pXA of the overexpression 5-Aminolevulinate synthase gene for building, obtains producing the glutamic acid of 5-ALA
Bar bacterium engineered strain L2；
Or PBP encoding gene pbp1b is knocked out in bacterial strain CB6, obtain bacterial strain CB8；Proceed in bacterial strain CB8
The plasmid pXA of the overexpression 5-Aminolevulinate synthase gene for building, obtains producing the glutamic acid of 5-ALA
Bar bacterium engineered strain L3；
Or PBP encoding gene pbp2b is knocked out in bacterial strain CB6, obtain bacterial strain CB9；Proceed in bacterial strain CB9
The plasmid pXA of the overexpression 5-Aminolevulinate synthase gene for building, obtains producing the glutamic acid of 5-ALA
Bar bacterium engineered strain L4.
2. the Corynebacterium glutamicum engineered strain of the production 5-ALA that the method described in claim 1 builds.
3. described in claim 2 production 5-ALA Corynebacterium glutamicum engineered strain purposes.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| CN114426983A (en) * | 2022-02-11 | 2022-05-03 | 天津大学 | Method for producing 5-aminolevulinic acid by knocking out transcription regulatory factor Ncgl0580 in corynebacterium glutamicum |
| CN116926102A (en) * | 2023-07-19 | 2023-10-24 | 天津大学 | Method for producing 5-ALA by inhibiting expression of global transcription regulatory factor gene glxR in corynebacterium glutamicum |
-
2016
- 2016-10-11 CN CN201610888618.5A patent/CN106636171A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| FENG L等: "Metabolic engineering of Corynebacterium glutamicum for efficient production of 5-aminolevulinic acid.", 《BIOTECHNOLOGY AND BIOENGINEERING》 * |
| ZHU N等: "Improved succinate production inCorynebacterium glutamicumby engineering glyoxylate pathway and succinate export system.", 《BIOTECHNOLOGY LETTERS》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| CN114426983A (en) * | 2022-02-11 | 2022-05-03 | 天津大学 | Method for producing 5-aminolevulinic acid by knocking out transcription regulatory factor Ncgl0580 in corynebacterium glutamicum |
| CN116926102A (en) * | 2023-07-19 | 2023-10-24 | 天津大学 | Method for producing 5-ALA by inhibiting expression of global transcription regulatory factor gene glxR in corynebacterium glutamicum |
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