CN104651289B - Acetic Acid Accumulation is to strengthen the genetic engineering bacterium and its construction method of L tryptophan yield in a kind of reduction fermentation process - Google Patents
Acetic Acid Accumulation is to strengthen the genetic engineering bacterium and its construction method of L tryptophan yield in a kind of reduction fermentation process Download PDFInfo
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Abstract
The invention discloses Acetic Acid Accumulation in a kind of reduction fermentation process to strengthen the genetic engineering bacterium and its construction method of L tryptophan yield, belong to metabolic engineering and genetic engineering field.The present invention is female parent with L tryptophan-producing Strains strain (recombination bacillus coli) FB 04 (being denoted as TS) phosphate based transferases (Pta), and rite-directed mutagenesis is carried out to it using Protocols in Molecular Biology.Under transforming herein, Pta Rate activities are 60.4% before mutation.Using Red recombinant techniques, pta genes on TS genomes are mutated by two steps seamless gene Shift Method, engineering bacteria TS PtaP69L are built.This strategy significantly reduces Pta Rate activity, and in fermentation process engineering bacteria TS PtaP69L compared with TS：1) shake flask fermentation tryptophane increase by 80%, acetic acid content reduces 15.5%；2) 3L ferment tanks tryptophane increase by 12.6%, acetic acid content reduces 73%.This tryptophan mutant strain has more preferable industrial fermentation performance.The genome rite-directed mutagenesis strategy can also be used for improving the fermenting property for producing other amino acid Escherichia coli.
Description
Technical field
The present invention relates to it is a kind of weaken fermentation process in Acetic Acid Accumulation with strengthen L-Trp yield genetic engineering bacterium and
Its construction method, the technology of Rate activity is reduced in particular with the directed mutagenesis method of protein engineering and Red restructuring is utilized
Technology, the technology of gene substitution is carried out by the seamless Shift Method of two steps on genome, belongs to metabolic engineering and genetic engineering neck
Domain.
Background technology
L-Trp (L-tryptophan, L-Trp) is widely used in doctor as a kind of essential amino acid in human body
The industries such as medicine, food, feed.In recent years, the external means by metabolic engineering, gradually cultivate the L-Trp lifes of a collection of high yield
Produce bacterial strain, but its still suffer from during the fermentation fermentation later stage L-Trp throughput rate decline it is obvious, the problems such as byproducts build-up,
The especially accumulation of by-product acetic acid.The growth of bacterial strain and L-Trp synthesis will be influenceed when acetic acid concentration is more than 2g/L, is
The accumulation of acetic acid in fermentation process is reduced as far as possible to ensure to must assure that dissolved oxygen fills in the normal growth of thalline, fermentation process
Foot, but this not only increases cost in industrialized production but also also is difficult to reach the dissolved oxygen condition that laboratory is provided.Therefore subtract
The accumulation of acetic acid and reduction thalline have important to the demand of dissolved oxygen to the industrialized production for improving L-Trp in few fermentation process
Meaning.
Escherichia coli generate acetic acid by two approach：(1) pyruvate oxidation enzymatic pathway, pyruvic acid is raw under PoxB effects
Into acetic acid；(2) acetyl phosphate approach, acetyl-CoA is in the lower generation acetyl-p of Pta (phosphate based transferase) effects, so
AckA (acetokinase) is catalyzed acetyl-p generation acetic acid afterwards, and the latter is the main path for generating acetic acid.Phosphate base turns
It is a key enzyme in microbial body in acetate metabolism approach AckA-pta to move enzyme, is also in industrial microorganism fermentation process
Produce one of key enzyme of by-product acetic acid.Therefore the phosphate based transferase mutant of Rate activity reduction is obtained, and to base
Because the original encoding gene in group is replaced, the phosphate based transferase of coding Rate activity reduction contributes to reduction to ferment
The accumulation of acetic acid in journey, so as to be advantageously implemented the high density fermentation of bacterial strain, and is conducive to increasing the generation of L-Trp.This
Invention with L-Trp production bacterial strain (recombination bacillus coli) FB-04 (being denoted as TS) (J Ind Microbiol Biotechnol,
2011,38:1921-1929) it is starting strain, by an alkali in phosphate based transferase encoding gene on its genome
Base is replaced, and causes its 69th proline encoded to be substituted for leucine, and the ratio for reducing phosphate based transferase is lived
The accumulation of by-product acetic acid is reduced in power, fermentation process, and the increase of L-Trp yield, is that industrial fermentation produces other amino acid
Offer reference.
The content of the invention
The invention provides Acetic Acid Accumulation in a kind of reduction fermentation process to strengthen the genetic engineering bacterium of L-Trp yield,
Characterized in that, in L-Trp production bacterial strain (recombination bacillus coli) FB-04 (being denoted as TS) (J Ind Microbiol
Biotechnol,2011,38:1921-1929) base in phosphate based transferase encoding gene is entered on genome
Row is replaced, and is caused its 69th proline encoded to be substituted for leucine, is reduced the Rate activity of phosphate based transferase, sends out
The accumulation of ferment byproduct in process thing acetic acid is reduced, the increase of L-Trp yield.
The phosphate based transferase mutant with Escherichia coli TS phosphate based transferases amino acid sequence (with
NCBI accession number:Amino acid sequence is identical shown in NP_416800.1) it is sequence of setting out, the 69th proline is substituted for bright ammonia
The mutant that acid is obtained.
The genetic engineering bacterium or transgenic cell line of phosphate based transferase mutant described in claim 1 can be expressed,
And the carrier of seamless replacement is carried out on genome fall within the claimed scope of this patent.
The present invention not only provides a kind of method for preparing the phosphate based transferase mutant, also provides one kind and prepares
The method of the mutant strain.Using Red recombination methods, gene substitution is carried out on genome by the seamless method of two steps.
Specifically, it is to set out sequence, inside it with Escherichia coli TS phosphate based transferases nucleotides sequence row
One base is replaced, and the 69th proline is substituted for leucine, and the progress phosphate group-transfer on TS genomes
The displacement of enzyme coding gene, makes its 69th proline of amino acid encoded be substituted for the mutant strain that leucine is obtained.
The Acetic Acid Accumulation for preparing in a kind of reduction fermentation process is to strengthen the side of the genetic engineering bacterium of L-Trp yield
Method, is comprised the following steps that：
1) phosphoric acid is obtained to being simulated from Escherichia coli phosphate based transferase using Swiss-model softwares
Acetyltransferase space structure, is analyzed by sequence and space structure to phosphate based transferase, it is determined that will
The amino acid sites of mutation.
2) according to Escherichia coli phosphate based transferase amino acid sequence (NCBI accession number:NP_416800.1), by it
Corresponding coding nucleotide sequence (the Genbank numbers of logging in NC_000913.3：2414747-2416891) it is cloned into plasmid
In pET-24a (+), construction recombination plasmid.
3) mutant primer is designed, rite-directed mutagenesis is carried out to phosphate based transferase gene order, by the ammonia in the site
Base acid is replaced, and obtains the recombinant vector containing mutation phosphate based transferase sequence.
4) overlapping PCR primers are designed, respectively with pKD13 plasmids and bacillus subtilis (Bacillus subtilis
Subsp.subtilis str.168) genome be masterplate, kanamycins and SacB gene phases are obtained by the method for over-lap PCR
DNA fragmentation even, this fragment is cloned into pMD18 carriers, the plasmid kan-sacB/pMD18 for seamless replacement is built.
5) the restructuring primer of phosphoric acid acetyl transferase gene homology arm is designed, using kan-sacB/pMD18 plasmids as mould
Version, obtains kan-sacB DNA fragmentation of the two ends containing homology arm using PCR method, this fragment is transferred in TS bacterium, Red is utilized
Restructuring obtains the mutant strain that phosphate based transferase gene is replaced by kan-sacB DNA fragmentations on genome.
6) using the recombinant vector of the 3) mutation as template, by the phosphate group-transfer of the mutation obtained with PCR method
Enzyme gene be transferred to 5) described in bacterial strain in, utilize Red restructuring obtain genome on phosphate based transferase mutation mutation
Bacterial strain.
The phosphate based transferase mutant Rate activity that the present invention is provided is significantly reduced, for 60.4% before mutation.Phase
For using the sieve means such as bacterium or mutagenesis, shortening the transformation time.By the phosphoric acid second on tryptophan-producing Strain strain TS genome
Acyltransferase encoding gene is substituted for the mutant being accordingly mutated, and obtains L-Trp mutant strain TS-PtaP69L.
The phosphate based transferase mutant is applied to industrial microorganism fermentation arts, is conducive in microorganism high density fermentation mistake
The accumulation of acetic acid is reduced in journey, increases the generation of fermentation purpose product, has broad application prospects.
Brief description of the drawings
Fig. 1 kan-sacB/pMD18 plasmid maps
Fig. 2 mutant strains (TS-PtaP69L) are with compareing (TS) shake flask fermentation acetic acid content
Fig. 3 mutant strains (TS-PtaP69L) are with compareing (TS) shake flask fermentation tryptophane
Fig. 4 mutant strains (TS-PtaP69L) are with compareing (TS) 3L ferment tank acetic acid contents
Fig. 5 mutant strains (TS-PtaP69L) are with compareing (TS) 3L ferment tank tryptophanes
Embodiment
Embodiment 1：The clone of wild type phosphate based transferase gene and the structure of recombinant bacterial strain
With phosphate based transferase coding gene sequence (the Genbank numbers of logging in NC_000913.3：2414747-
2416891) it is template, designs amplimer P1 and P2, phosphate based transferase gene pta, clone is expanded using PCR method
To pMD18 carriers (commercialization tool carrier), connection product conversion e. coli jm109, converted product coating ammonia containing 100mg/L
The LB flat boards of parasiticin.Through 37 DEG C of overnight incubations, choosing colony extracts plasmid after accessing LB fluid nutrient mediums, 8-10h, ordered
Entitled pta/pMD18, sequencing is carried out by this plasmid.As a result DNA fragmentation of the Insert Fragment for a 2145bp, coding are shown
One protein containing 704 amino acid.Carrier T by pET24a (+) plasmid and containing pta genes carries out the Hes of Nde I respectively
The double digestions of Hind III, digestion products are tapped rubber after recovery, then are connected with T4 ligases.Connection product Transformed E .coli JM109 experience
State cell, through 37 DEG C culture 8h, choose transformant received containing 30mg/L cards mycin LB in shaken cultivation, extraction plasmid, digestion
Checking, obtains expression plasmid pta/pET24a (+).
By plasmid pta/pET24a (+) Transformed E .coli BL21 (DE3) Host Strains, coating receives mycin (30mg/L) containing card
LB flat boards on, 37 DEG C culture 8h, be named as pta/pET24a (+)/BL21 (DE3).Choose single bacterium to drop down onto in liquid LB, 37 DEG C of trainings
Support overnight, preserve glycerol tube.
Phosphate based transferase encoding gene amplimer：
Forward primer P1：5’-AAACATATGTCCCGTATTATTATGCTGATCC-3’
Reverse primer P2：5’-CCCAAGCTTTTACTGCTGCTGTGCAGACTG-3’
Embodiment 2：The preparation of mutant
1) structure of mutant
To from L-Trp production bacterial strain (recombination bacillus coli) FB-04 (being denoted as TS) (J Ind Microbiol
Biotechnol,2011,38:1921-1929) phosphate based transferase protein structure is simulated, it was found that phosphoric acid second
The amino acid sites Pro69 that one has potential impact to the aggressiveness stability of enzyme six in acyltransferase molecule.By phosphoric acid second
The proline (Pro) of the 69th has been mutated into leucine (Leu) in acyltransferase, is named as P69L.According to phosphate base
Transferase coding gene sequence (the Genbank numbers of logging in NC_000913.3：2414747-2416891), introducing is designed and synthesized
The primer R1 and R2 of P69L mutation, with plasmid pta/pET24a (+) for template, are pinpointed to phosphate based transferase gene
Mutation.PCR primer digests through Dpn I (Fermentas companies), converts escherichia coli jm109 competent cell, competent cell
After LB solid mediums (received containing 30 μ g/mL cards mycin) overnight incubation, choose monoclonal and (contain 30 μ g/mL in LB fluid nutrient mediums
Card receives mycin) in cultivate, plasmid is extracted afterwards, mutant plasmid translation table is reached into host e. coli BL21 (DE3) competent cell,
Mutant plasmid sequencing is correct, determines DNA encoding sequence, identifies the mutant that the 69th Pro codon becomes Leu codons.
Introduce P69L mutation rite-directed mutagenesis primer be：
Forward primer R1：5’-GACGGCCGCTGAACTTCTGCTGAAAATGAG-3’
Reverse primer R2：5’-CTCATTTTCAGCAAGAGTTCAGCGGCCGTC-3’
2) expression and purification of mutant enzyme
Picking positive monoclonal is in LB fluid nutrient mediums (receiving mycin containing 30 μ g/mL cards) 8~10h of growth, by 5% inoculum concentration
Seed fermentation liquid is connected to TB fluid nutrient mediums (receiving mycin containing 30 μ g/mL cards)；Escherichia coli are in 37 DEG C of shaking table cultures to OD600
=0.6, the IPTG induction extracellular expressions of 0.4mM final concentrations are added, and after 25 DEG C of shaking tables continue cultivation and fermentation 24h, will ferment
Liquid removes supernatant in 4 DEG C, 10000g centrifugations 10min, collects thalline.
Use 20mmol KHCO3By mycelium dilution to OD600Broken wall after being approximately equal to 20,4 DEG C, 10000g centrifugation 30min go to sink
Form sediment, collect supernatant.It is slowly added to protect (the NH of sum into supernatant4)2SO4Solution is protected and spent to 50%, 4 DEG C of placement 2h.4℃、
10000g centrifuges 30min, collects precipitation.Redissolved and precipitated with 50mmol/L pH=8.0Tris-Hcl buffer solutions, containing 0.2mol/
L(NH4)2SO410mmol/L pH=7.6 Tris-Hcl buffer solutions in dialysed overnight, during which change 2-3 dialysis buffer
Liquid, by stand-by after 0.22 μm of membrane filtration.
The enzyme liquid after filtering is diluted 4 times with 10mmol/L pH=8.5 Tris-Hcl buffer solutions, using AKTA albumen
The purifying that instrument carries out recombinant protein is purified, whole process of purification is carried out in chromatography cabinet, and it is 4 DEG C to control temperature.Anion exchange
Chromatographic purification step：(1) balance：With (the NH containing 0.05mol/L of 5 times of volumes4)2SO410mmol/L pH=7.8 Tris-
Hcl buffer solutions balance DEAE anion-exchange columns；(2) loading：The good sample of beforehand dilution is with 1mL/min flow velocity
Sample；(4) elute, flow velocity 1.0mL/min, with containing 0.2mol/L (NH4)2SO410mmol/L pH=7.8 Tris-Hcl buffering
Liquid carries out gradient elution, and Detection wavelength is 280nm, the eluent of fraction collection phosphoric acid acetyltransferase, -20 DEG C of preservations.
Embodiment 3：The enzyme activity of DTNB determination of color phosphate based transferases
1) enzyme activity determination method
The enzyme activity of phosphate based transferase uses the thiobis of 5,5'- bis- (2- nitrobenzoic acids) (DTNB) development process.
Under the conditions of 25 DEG C, phosphate based transferase catalysis substrate acetyl coacetylase generation coacetylase, coacetylase and DTNB react generation
A kind of yellow substance, there is absorbance at wavelength 412nm, and the depth of its color and the amount of coacetylase are into just within the specific limits
Than, therefore colorimetric can be carried out under 412nm wavelength, calculate enzyme activity.Enzyme-activity unit is defined：Under these conditions, it is per minute to urge
The enzyme amount for changing 1 μm of ol coacetylase of generation is used as a unit of activity.
Enzyme activity determination step：Under the conditions of 25 DEG C, 850ml 0.1mol/L pH=7.4 are sequentially added in 1ml cuvettes
Phosphate buffer, 50ml 1.6mmol/L DTNB, 50ml 4mmol/L acetyl coenzyme A (DTNB solution and acetyl coenzyme A
Solution is all configured with 0.1mol/L pH=7.4 phosphate buffer).It is eventually adding 50ml phosphate based transferase enzyme liquids anti-
2min is answered, its light absorption value is measured under 412nm and enzyme activity is calculated.
2) specific enzyme activity is compared：
Experimental result is listed in table 1, by the pure enzyme of mutant compared with the pure enzyme of wild type, it can be found that：The ratio of mutant enzyme is lived
Power is significantly reduced, and is 1250.1U/mg than living, is the 60.4% of wild specific activity of enzyme.
The Rate activity of the wild enzyme of table 1 and mutant enzyme
Embodiment 4：The displacement of phosphate based transferase gene in tryptophan-producing Strain pnca gene group
Overlapping PCR primers are designed, respectively using pKD13 plasmids and Bacillus subtilis genes group as masterplate, pass through over-lap PCR
Method obtains the DNA fragmentation that kanamycins is connected with SacB genes, and this DNA fragmentation is cloned into pMD18 carriers, builds plasmid
kan-sacB/pMD18.The restructuring primer of phosphoric acid acetyl transferase gene homology arm is designed, with kan-sacB/pMD18 matter
Grain is masterplate, obtains kan-sacB DNA fragmentation of the two ends containing homology arm using PCR method, ethanol precipitation precipitates this DNA piece
Section, 12000g centrifugation 10min remove supernatant, -20 DEG C save backup after the redissolution of 10ul deionized waters.It is mutated with same procedure
Phosphate based transferase gene afterwards, -20 DEG C save backup.
Overlapping PCR primers：
Kan-F；5’-AAACATATGATTCCGGGGATCCGTCGACC-3’
SacB-R；5’-CCCAAGCTTTGCCAATAGGATATCGGCAT-3’
Overlapping-F；5’-CGAAGCAGCTCCAGCCTACAGCAACTTTATGCCCATGCA-3’
Overlapping-R；5’-TGCATGGGCATAAAGTTGCTGTAGGCTGGAGCTGCTTCG-3’
Restructuring primer containing homology arm：
Homologous-F；5’-GTGTCCCGTATTATTATGCTGATCCCTACCGGAACCAGCG
TCGGTCTGACATTCCGGGGATCCGTCGACC-3’
Homologous-R；5’-TTACTGCTGCTGTGCAGACTGAATCGCAGTCAGCGCGATG
GTGTAGACGATGCCAATAGGATATCGGCAT-3’
The phosphate based transferase gene magnification primer of mutation：
F；5’-GTGTCCCGTATTATTATGCTGATCCC-3’
R；5’-TTACTGCTGCTGTGCAGACTGAATC-3’
Former L-Trp production engineering bacterium TS is prepared into competent cell first, plasmid pKD46 and TS competence is thin
Born of the same parents are mixed, and electroporated, add 37 DEG C of culture 2h after appropriate amount of fluid LB, and LB of the coating containing ampicillin (100mg/L) is put down
On plate, 30 DEG C of culture 10-16h choose single bacterium and dropped down onto in liquid LB, 30 DEG C of overnight incubations preserve glycerol tube.This is contained into pKD46
The TS bacterium of plasmid are transferred in the liquid LB containing ampicillin (100mg/L) and kanamycins (30mg/L), 30 DEG C of culture 12h
Afterwards, it is transferred in SOB culture mediums, is induced with final concentration 30mM arabinoses after 4h by 10% inoculum concentration, is prepared into electricity conversion sense
By state cell.Competent cell is mixed with the kan-sacB DNA fragmentations containing homology arm, after electroporated and incubation, coating contains
On the LB flat boards of ampicillin (100mg/L) and kanamycins (30mg/L), 30 DEG C of culture 10-16h.Single bacterium is chosen to drop down onto containing ammonia
In the liquid LB of parasiticin (100mg/L) and kanamycins (30mg/L), 30 DEG C of overnight incubations, PCR checkings preserve glycerine
Pipe.PCR is verified into correct single bacterium colony, competence is prepared into by identical method, electrotransformation is transferred to the phosphate of mutation
On based transferase gene, LB flat boards of the coating containing 15% sucrose, 37 DEG C of culture 10h.Choose the liquid that single bacterium drops down onto 15% sucrose
In LB, 37 DEG C of overnight incubations, PCR checkings preserve glycerol tube, obtain the tryptophan of phosphate based transferase mutation on genome
Bacterial strain is produced, its 69th proline of amino acid encoded is substituted for leucine, the chromogenic Histidine mutations bacterial strain successfully constructed
It is named as TS-PtaP69L.
Embodiment 5：Primary colors propylhomoserin produces bacterial strain and mutant strain shake flask fermentation result
By mutant strain (TS-PtaP69L) with compareing in (TS) 37 DEG C of shake flask fermentation 48h, fermentation process every 8h use
The regulation of 20% ammoniacal liquor makes medium pH maintain 6.5-7.2.Shake flat experiment result such as Fig. 2 and Fig. 3, mutant strain (TS-
PtaP69L) compared with control is (before mutation), it can be found that：1) final L-Trp content：It is 1.16g/l to compare (TS), and is dashed forward
Become bacterial strain (TS-PtaP69L) into 2.09g/l, compared with the control tryptophane increase by 80.7%；2) content of final acetic acid：
It is 12.9g/l to compare (TS), and mutant strain (TS-PtaP69L) is only 10.9g/l, and acetic acid content is reduced compared with the control
15.5%.
Fermentation medium (g/L)：Dipotassium hydrogen phosphate 24, potassium dihydrogen phosphate 9.6, epsom salt l, ammonium sulfate 5, glucose
30, dusty yeast 15, Citric Acid Mono 2, liquid microelement 3mI/L.
Liquid microelement (g/L)：Al2(SO4)3·18H2O 2, CoSO4·7H2O 0.75, CuS04·5H2O25,
H3BO30.5, MnSO4·H2O24, Na2MOO4·2H2O 3, NiSO4·6H2O 2.5, ZnSO4·7H2O 15, FeSO4·
7H2O25。
Embodiment 6：Primary colors propylhomoserin produces bacterial strain and mutant strain 3L ferment tank results
Bacterium solution is drawn from mutant strain (TS-PtaP69L) and control (TS) glycerol tube respectively, is connect by 0.2% inoculum concentration
Enter in seed culture medium, after 37 DEG C of culture 10h, accessed by 10% inoculum concentration in 3L fermentation tanks.Whole fermentation processes temperature
For 37 DEG C, 20% ammoniacal liquor maintains medium pH to be 6.5.It is 20% that dissolved oxygen is maintained before dissolved oxygen bounce-back, by 0.15 after solution bounce-back
Specific growth rate stream adds 800g/l glucose solution, and maintains dissolved oxygen to be 15%, fermentation to 58h.3L ferment tank results
Such as Fig. 4 and Fig. 5, mutant strain (TS-PtaP69L) compared with compareing (before mutation), it can be found that：1) final L-Trp contains
Amount：It is 39.6g/l to compare (TS), and mutant strain (TS-PtaP69L) is 44.6g/l, compared with the control tryptophane increase
12.6%；2) content of final acetic acid：It is 2.6g/l to compare (TS), and mutant strain (TS-PtaP69L) is only 0.7g/l, with
Control reduces 73% compared to acetic acid content.
Seed culture medium (g/L)：Dipotassium hydrogen phosphate 24, potassium dihydrogen phosphate 9.6, epsom salt l, ammonium sulfate 5, glucose
30, dusty yeast 15.
Fermentation medium (g/L)：Dipotassium hydrogen phosphate 15, potassium dihydrogen phosphate 9.6, epsom salt 2, ammonium sulfate 1.6, grape
Sugar 7.5, dusty yeast 2, Citric Acid Mono 2, liquid microelement 3mI/L (identical with liquid microelement in shaking flask).
Claims (6)
1. Acetic Acid Accumulation is to strengthen the genetic engineering bacterium of L- tryptophan yield in a kind of reduction fermentation process, it is characterised in that
By a base in phosphate based transferase encoding gene on L- tryptophan-producing Strains strain Escherichia coli TS genomes
It is replaced, causes its 69th proline encoded to be substituted for leucine, the ratio for reducing phosphate based transferase is lived
The accumulation of by-product acetic acid is reduced in power, fermentation process, the increase of L- tryptophans yield.
2. a kind of phosphate based transferase mutant, it is characterised in that be with Escherichia coli TS phosphate based transferases
Amino acid sequence is sequence of setting out, and the 69th proline is substituted for into the mutant that leucine is obtained, the Escherichia coli TS
The amino acid sequence of phosphate based transferase is NCBI accession number:Amino acid sequence shown in NP_416800.1.
3. the construction method of genetic engineering bacterium described in claim 1, it is characterised in that phosphate base is carried out on genome
The displacement of enzyme coding gene is shifted, its 69th proline of amino acid encoded is substituted for leucine and obtains mutant strain.
4. the construction method according to claim 3, it is characterised in that comprise the following steps that：1) Swiss- is utilized
Model softwares are simulated to the phosphate based transferase from Escherichia coli TS, obtain phosphate based transferase space
Structure, is analyzed by sequence and space structure to phosphate based transferase, it is determined that the amino acid sites to be mutated；
2) according to Escherichia coli TS phosphate based transferase amino acid sequences, by the coding nucleotide sequence gram corresponding to it
It is grand to arrive in plasmid pET-24a (+), construction recombination plasmid；
3) mutant primer is designed, rite-directed mutagenesis is carried out to phosphate based transferase gene order, by the amino acid in the site
It is replaced, obtains the recombinant vector containing mutation phosphate based transferase sequence；
4) overlapping PCR primers are designed, respectively with pKD13 plasmids and bacillus subtilis (Bacillus subtilis subsp.
Subtilis str.168) genome be masterplate, kanamycins and SacB gene phases are obtained by the method for over-lap PCR
DNA fragments even, this fragment is cloned into pMD18 carriers, the plasmid kan-sacB/ for seamless replacement is built
pMD18；
5) the restructuring primer of phosphoric acid acetyl transferase gene homology arm is designed, using kan-sacB/pMD18 plasmids as mould
Version, obtains kan-sacB DNA fragment of the two ends containing homology arm using PCR methods, this fragment is transferred in TS bacterium,
Recombinated using Red and obtain the mutant bacteria that phosphate based transferase gene is replaced by kan-sacB DNA fragments on genome
Strain；
6) using the recombinant vector of the 3) mutation as template, by the phosphate group-transfer of the mutation obtained with PCR methods
Enzyme gene be transferred to 5) described in bacterial strain in, utilize Red restructuring obtain genome on phosphate based transferase mutation
Mutant strain.
5. phosphate based transferase mutant carries out seamless replacement on genome described in energy Expression product claim 2
Plasmid.
6. Acetic Acid Accumulation in fermentation process is weakened described in claim 1 with the genetic engineering bacterium for strengthening L- tryptophan yield to exist
The application of other amino acid of fermenting and producing and protein field.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101023178A (en) * | 2004-09-17 | 2007-08-22 | 莱斯大学 | High succinate producing bacteria |
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| CN101023178A (en) * | 2004-09-17 | 2007-08-22 | 莱斯大学 | High succinate producing bacteria |
Non-Patent Citations (3)
| Title |
|---|
| "In Vivo and in Vitro Analyses of Single-amino Acid Variants of the Salmonella enterica Phosphotransacetylase Enzyme Provide Insights into the Function of Its N-terminal Domain";Shaun R. Brinsmade et al.;《THE JOURNAL OF BIOLOGICAL CHEMISTRY》;20070227;第282卷(第17期);摘要，第12630、12631和12635页 * |
| "L-色氨酸工程菌的改造及发酵条件控制";郭宝珠;《中国优秀硕士学位论文全文数据库工程科技I辑》;20140915(第9期);摘要，第2.2节、2.4节和第54页 * |
| Accession Number： NP_416800.1，phosphate acetyltransferase[Escherichia coli str.K-12 substr.MG1655];Riley, M., et al.;《GenBank》;20101014;全文 * |
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