CN108753860A - The structure of Recombinant organism and its purposes of production L-Trp - Google Patents
The structure of Recombinant organism and its purposes of production L-Trp Download PDFInfo
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Abstract
The present invention relates to one plant of Recombinant organism and the methods for utilizing its fast and efficient production L-Trp.The genetic engineering bacterium is to carry out corresponding transformation to L-Trp synthesis related gene in Escherichia coli by the method (including gene integration and point mutation are replaced with promoter) of metabolic engineering to combine, and obtain after glutamine synthelase encoding gene glnA of the introducing from lactobacillus acidophilus on its genome.10-12g/L can be reached in interior accumulation L-Trp for 24 hours by carrying out shake flask fermentation using the bacterial strain, for the peak of country's report at present；The bacterial strain can accumulate L-Trp in 5L ferment tanks 35-40h and reach 40-45g/L, improve 15.1% compared to the existing L-Trp industrialization bacterial strain for carrying plasmid, illustrate that the bacterial strain has the potentiality of good industrialized production of L-tryptophan.
Description
Technical field
The present invention relates to Recombinant organism and its construction methods, and utilize the engineering bacteria fast and efficient production
The method of L-Trp, belongs to microorganism metabolism controlling and gene engineering technology field.
Background technology
L-Trp is a kind of essential amino acid, be widely used as feed addictive, drug such as infusion solution and
The basic material of health functional food etc..Its production method includes chemical synthesis, enzyme reaction method, fermentation method etc., but main at present
The direct fermentation using microorganism, this method is used to pass through fermenting and producing L- color ammonia by substrate of low price raw materials such as glucose
Acid, production cost is relatively low, and production technology is relatively easy controllable.
Since the metabolic pathway of synthesizing of L-Trp is long and complex, involved precursor is more, in the large intestine of wild type
The level that L-Trp is accumulated in bacillus is extremely low.Initial research mainly builds L-Trp production by way of mutation breeding
Bacterial strain obtains many and relieves feedback inhibition then by the way that mutagenesis obtained strains are carried out with the sequencing of some key genes
Key gene sequence.In conjunction with genetic engineering means, the obtained key gene for releasing feedback inhibition is overexpressed
It is the main direction of studying for building Escherichia coli L-Trp engineered strain to strengthen the route of synthesis of L-Trp.
In its oligogene engineering means, the overexpression mode for playing the key gene of key effect is typically chosen and passes through
Structure is overexpressed plasmid mode and carries out, and plasmid all has many characteristics, such as strong promoter, high copy number mostly.Though the overexpression mode
It can so achieve the purpose that strengthen L-Trp route of synthesis, but there is also carry resistance, need to add for constructed engineered strain
Derivant brings the adverse effects such as burden to thalli growth.Chen L and Zeng AP (Applied Microbiology&
Biotechnology.2017.101(2):559) the Escherichia coli L- for not carrying plasmid is constructed by the method for gene integration
Tryptophan engineering bacteria analyzes the outer intermediate product of intracellular and precursor variation in its fermentation process, is mentioned after fermentation in text
Phase intracellular L-Glutamine content is relatively low, and the precursor as synthesis L-Trp can directly affect fermenting and producing L-Trp
Yield and conversion ratio.
The regeneration of L-Glutamine is to be catalyzed Pidolidone and ammonium salt by glutamine synthelase in Bacillus coli cells
It reacts to complete.However, adenylylation phenomenon can occur for glutamine synthelase, its enzyme activity is caused to drastically reduce.In ammonium salt
When more than demand, adenylylation degree is reinforced, and activity of glutamine synthetase, which declines, even to be inactivated, but the life of L-Glutamine
At indispensable ammonium salt supply again.In addition, in the fermentation process such as amino acid, organic acid, since intracellular product accumulation leads to pH
Decline, the enzyme activity of conventional microbiological glutamine synthelase is relatively low at this time.Therefore, seek high activity glutamine synthelase to come by force
The regeneration for changing intracellular L-Glutamine has important application value in the products such as biosynthesis L-Trp.
Currently, the domestic Escherichia coli for producing L-Trp are the bacterial strain for carrying plasmid mostly, matter in fermentation process
Grain it is easy to be lost, in addition the precursor that L-Trp route of synthesis is related to it is more, fermentation the later stage easily accumulate more by-product etc. because
There is difficult control, yield and saccharic acid and convert in the influence of element, the zymotechnique of existing L-Trp colibacillus engineering strain
The problems such as rate is relatively low, extraction process is complicated.
Invention content
For above-mentioned there are problem, purpose of the present invention is to the method structures by metabolic engineering and genetic engineering can be efficient
The Recombinant organism for producing L-Trp, to realize the safe, fast, efficient production of L-Trp.The base
Because engineering bacteria is the method (including gene integration and point mutation are replaced with promoter) by metabolic engineering, to the L- of Escherichia coli
Related gene is combined obtained from transformation in tryptophan synthetic pathway.Relevant base is synthesized in Escherichia coli with L-Trp
Because including that (3-phoshoglyceric acid is de- by aroG (3- deoxidations-σ-Arab's heptanone saccharic acid -7- phosphate synthases encoding gene), serA
Hydrogen enzyme coding gene), trpEDCBA (tryptophan operon encoding gene).
It uses and such as gives a definition in the present invention：
1, the nomenclature of amino acid and DNA nucleic acid sequences
Using the generally acknowledged IUPAC nomenclatures of amino acid residue, with a letter abbreviations form.DNA nucleic acid sequences are using generally acknowledged
IUPAC nomenclatures.
2, the mark of mutant
The amino acid being mutated in mutant is indicated using " amino acid that Original amino acid position is replaced ".Such as trpE
(S40F), indicate that the amino acid of the amino acid sequence positions 40 of trpE coded by said gene is substituted for benzene by the serine (S) of parent
Alanine (F).Such as serA (H344A, N364A), indicate that the amino acid of position 344 and position 364 is all mutated.
Technical scheme of the present invention is summarized as follows：
One of technical scheme of the present invention：It is the genetic engineering bacterium for building one plant of energy fast and efficient production L-Trp
E.coli TRP03, the genetic engineering bacterium are to replace the promoter of tryptophan operon on the genome of Escherichia coli
It is mutated to introduce trpE (S40F) while Ptrc promoters；AroG (S180F) gene integration that will be controlled by Ptrc promoters
To the sites tyrR；SerA (H344A, the N364A) gene integrations that Plac promoters are controlled are to yjiV pseudogenes site.
The starting strain of the Escherichia coli is preferably E.coli W3110 (number ATCC 27325).
The construction step of said gene engineering bacteria is summarized as follows：
(1) Escherichia coli CRISPR/Cas gene editing technologies are used, using E.coli W3110 as starting strain, by color ammonia
The promoter of sour operon introduces trpE (S40F) gene of solution feedback inhibition while replacing with Ptrc promoters, strengthen color ammonia
The feedback inhibition of key enzyme anthranilate synthase is released while sour operon synthetase series；
(2) aroG (S180F) gene integrations for the solution feedback inhibition for controlling Ptrc promoters are strengthened and are closed to the sites tyrR
Its feedback inhibition is released while key enzyme 3- deoxy-arabinose heptanone saccharic acid -7- phosphate synthases；
(3) serA (H344A, the N364A) gene integrations controlled Plac promoters are to yjiV pseudogenes site, before increase
Body object Serine releases its feedback inhibition while synthesis.
The nucleotide sequence of the Ptrc promoters such as SEQ ID No：Shown in 1.
The nucleotide sequence such as SEQ ID No of trpE (S40F) gene：Shown in 2.
The nucleotide sequence such as SEQ ID No of aroG (S180F) gene：Shown in 3.
The nucleotide sequence of the Plac promoters such as SEQ ID No：Shown in 4.
The nucleotide sequence such as SEQ ID No of serA (H344A, the N364A) gene：Shown in 5.
The nucleotide sequence of the tyrR genes such as SEQ ID No：Shown in 6.
The nucleotide sequence of the yjiV genes such as SEQ ID No：Shown in 7.
The two of technical scheme of the present invention：It is the genetic engineering bacterium of one plant of fast and efficient production L-Trp of further structure
E.coli TRP04, the genetic engineering bacterium are introduced on the basis of genetically engineered E.coli TRP03 from acidophilus breast
The glutamine synthelase encoding gene glnA of bacillus (Lactobacillus acidophilus)；Further, it is by Plac
(the front and back nucleotide sequence of optimization is respectively such as SEQ ID No for glnA genes after the optimization of promoter control：8 and SEQ ID
No：Shown in 9) it is integrated into the ycjV pseudogenes site (nucleotide sequence of the ycjV genes such as SEQ ID No：Shown in 10), increase
Add the supply of precursor L-Glutamine.
The three of technical scheme of the present invention：It is to utilize application of the said gene engineering bacteria in fermenting and producing L-Trp.
It is as follows that shake flask fermentation is carried out using said gene engineering bacteria：
Seed liquor will be prepared after actication of culture, the 500mL triangles equipped with fermentation medium are inoculated by 10-15% inoculum concentrations
In bottle, nine layers of gauze seal, 37 DEG C, 200r/min shaken cultivations, maintain pH in 7.0- by adding ammonium hydroxide in fermentation process
7.2；Adding 60% (m/v) glucose solution maintains fermentation (give instruction agent with phenol red, when zymotic fluid color no longer changes
It is considered as and lacks sugar, 1-2mL60% (m/v) glucose solution is added when lacking sugar)；Fermentation period 20-24h.
Preferred fermentation medium group becomes：Glucose 20-40g/L, (NH4)2SO42-6g/L, KH2PO41-5g/L,
MgSO4·7H2O 0.5-2g/L, yeast extract 1-5g/L, FeSO4·7H2O 30-60mg/L, MnSO4·7H2O 1-5mg/
L, VH0.1-0.5mg/L, VB10.5-1.0mg/L, micro-mixed liquor 1-3ml/L, phenol red 15-30g/L, remaining is
Water, pH 7.0-7.2,115 DEG C of high steam pots sterilizing 15min；
Micro-mixed liquor group is divided into：Na2MoO4·2H2O 2.5g/L, AlCl3·6H2O 2.5g/L, NiSO4·
6H2O 2.5g/L, CoCl2·6H2O 1.75g/L, CaCl2·2H2O 10g/L, ZnSO4·7H2O 0.5g/L, CuCl2·2H2O
0.25g/L, H3BO3 0.125g/L。
The yield of L-Trp is up to 8-9g/L after engineering strain E.coli TRP03 shake flask fermentations 20-24h,
The yield of L-Trp is up to 10-12g/L after E.coli TRP04 shake flask fermentations 20-24h.
It is as follows that 5L ferment tanks are carried out using said gene engineering bacteria：
Seed liquor will be prepared after actication of culture, fresh fermentation medium is accessed according to 15-20% inoculum concentrations, starts to send out
Ferment, control pH stablizes 7.0 or so in fermentation process, and temperature maintains 37 DEG C, and dissolved oxygen is between 25-35%；When in culture medium
Glucose consumption it is complete after, stream plus 80% (m/v) glucose solution, maintain fermentation medium in concentration of glucose exist
0.1-5g/L；Fermentation period 35-40h；
Preferred fermentation medium group becomes：Glucose 20-40g/L, (NH4)2SO42-6g/L, KH2PO41-5g/L,
MgSO4·7H2O 0.5-2g/L, yeast extract 1-5g/L, FeSO4·7H2O 30-60mg/L, MnSO4·7H2O 1-5mg/
L, VH0.1-0.5mg/L, VB10.5-1.0mg/L, micro-mixed liquor 1-3ml/L, remaining is water, pH 7.0-7.2,115
DEG C high steam pot sterilizes 15min；
Micro-mixed liquor group is divided into：Na2MoO4·2H2O 2.5g/L, AlCl3·6H2O 2.5g/L, NiSO4·
6H2O 2.5g/L, CoCl2·6H2O 1.75g/L, CaCl2·2H2O 10g/L, ZnSO4·7H2O 0.5g/L, CuCl2·2H2O
0.25g/L, H3BO3 0.125g/L。
Genetically engineered E.coli TRP04 can accumulate L-Trp using 5L ferment tanks 35-40h and reach 40-45g/L.
The invention has the advantages that and advantageous effect：
Plasmid is not carried in L-Trp engineered strain fermentation process provided by the invention, any antibiotic is not added and lures
Agent is led, brings facility for safety in production L-Trp, and the bacterial strain genetic background is clear, genetic manipulation is simple and efficient, convenient
Further character improvement is carried out to it；It is provided by the invention to combine and introduced from lactobacillus acidophilus through metabolic engineering
The L-Trp engineered strain of glutamine synthetase gene not only can effectively accumulate L-Trp, and be apparently higher than existing
The industrialization L-Trp for carrying plasmid produces bacterial strain, and shake flask fermentation produces L-Trp in for 24 hours and reaches 10-12g/L, for domestic institute
The peak of report；The present invention introduces L- color ammonia after being optimized from the glutamine synthetase gene of lactobacillus acidophilus
Sour engineered strain considerably improves the yield of L-Trp, and the gene is relevant applied to L-Glutamine synthesis is related to
The structure of Recombinant organism increases the yield of purpose product, has broad application prospects；L- provided by the invention
Tryptophan engineered strain can accumulate L-Trp 44.2g/L in 5L ferment tanks 40h, compared to the industrialization L- colors for carrying plasmid
Propylhomoserin production bacterial strain improves 15.1%, shows the potentiality of industrialized production of L-tryptophan.
Description of the drawings
Fig. 1：(a) pREDCas9 plasmid maps, (b) pGRB plasmid maps.
Fig. 2：Ptrc trpE (S40F) gene replaces segment structure and verification electrophoretogram.Wherein：M：1kb DNA
marker；1：Upstream homology arm；2：TrpE (S40F) and downstream homology arm；3：Overlapping fragments；4：Positive bacteria identifies segment；5：It is former
Bacterium compares.
Fig. 3：Ptrc aroG (S180F) gene integration segment is built and verification electrophoretogram.Wherein：M：1kb DNA
marker；1：Upstream homology arm；2：AroG (S180F) genetic fragment；3 downstream homology arms；4：Overlapping fragments；5：Opportunistic pathogen compares；
6：Positive bacteria identifies segment.
Fig. 4：Plac serA (H344A, N364A) gene integration segment is built and verification electrophoretogram.Wherein：M：1kb DNA
marker；1：Upstream homology arm；2：SerA (H344A, N364A) genetic fragment；3 downstream homology arms；4：Overlapping fragments；5：Opportunistic pathogen
Control；6：Positive bacteria identifies segment.
Fig. 5：Plac glnA gene integration segments are built and verification electrophoretogram.Wherein：M：1kb DNA marker；1：On
Swim homology arm；2：GlnA genetic fragments；3 downstream homology arms；4：Overlapping fragments；5：Opportunistic pathogen compares；6：Positive bacteria identifies segment.
Fig. 6：HPLC measures L-Trp standard curve.
Fig. 7：Different strains shake flask fermentation L-Trp yield.
Fig. 8：5L fermentation tank L-Trp resultant curves.
Specific implementation mode
The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention
It is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, it is not intended to limit the present invention
Range, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this
Under the premise of invention spirit and scope, various changes or change to material component and dosage progress in these embodiments
It belongs to the scope of protection of the present invention.
The percentage sign " % " arrived involved in embodiment refers to percent by volume if not specified；The percentage of solution
" % (m/v) " refers to the grams containing solute in 100mL solution.
Embodiment 1：Build Escherichia coli L-Trp engineered strain E.coli TRP03
1. the method for gene editing
Gene editing method reference literature (Li Y, Lin Z, Huang C, the et al.Metabolic used in the present invention
engineering of Escherichia coli using CRISPR–Cas9meditated genome
editing.Metabolic engineering,2015,31:It 13-21.) carries out, two plasmid maps used in this method are shown in
Attached drawing 1.Wherein pREDCas9 carries the elimination system of gRNA expression plasmids pGRB, the Red recombination systems and Cas9 eggs of bacteriophage lambda
White expression system, miramycin resistance (working concentration：100mg/L), 32 DEG C of cultures；PGRB is using pUC18 as skeleton, including promoter
J23100, gRNA-Cas9 calmodulin binding domain CaM sequence and terminator sequence, amicillin resistance (working concentration：100mg/L), 37
DEG C culture.
This method is as follows：
1.1 pGRB plasmid constructions
The purpose of structure plasmid pGRB be in order to transcribe corresponding gRNA, to the complex formed with Cas9 albumen, and
By base pairing and PAM identifying purpose gene target sites, target DNA double-strand break is realized.Using the DNA pieces comprising target sequence
Section and the method for the carrier segments recombination of linearisation build pGRB plasmids.
1.1.1 target sequence designs
Use CRISPR RGEN Tools design target sequences (PAM:5'-NGG-3')
1.1.2 include the preparation of the DNA fragmentation of target sequence
Design primer：5 '-linearized vector end sequences (15bp)-restriction enzyme site-target sequence (not including PAM sequences)-
The primer of linearized vector end sequence (15bp) -3 ' and its reverse complemental, it includes target sequence to be prepared by the annealing of single stranded DNA
DNA fragmentation.Reaction condition：95 DEG C of pre-degeneration, 5min；30-50 DEG C of annealing, 1min.Annealing system is as follows：
Annealing system
1.1.3 the preparation of linear carrier
The method that the linearisation of carrier uses Inverse PCR amplification.
1.1.4 recombining reaction
The following table of recombination system.Recombinase used isII One Step Cloning Kit series
Enzyme, recombinate condition：37 DEG C, 30min.
Recombination system
1.1.5 the conversion of plasmid
10 μ L reaction solutions are taken, 100mL DH5 αization is added to and turns in competent cell, gently ice bath 20min after mixing, 42
DEG C heat shock 45-90s, ice bath 2-3min, is added 900 μ L SOC, in 37 DEG C of recovery 1h immediately.8000rpm centrifuges 2min, abandons part
Supernatant is applied to the tablet containing 100mg/L ampicillins after staying 200 μ L or so that thalline is resuspended, tablet is inverted, in 37
It DEG C is incubated overnight.It is identified by bacterium colony PCR after tablet grows single bacterium colony, selects positive recombinant.
1.1.6 clone identification
PCR positive bacterium colonies are seeded in the LB culture mediums containing 100mg/L ampicillins after being incubated overnight and protect bacterium, carried
Take plasmid, digestion identification.
The preparation of 1.2 recombinant dna fragments
Recombinant fragment for knockout is made of that (upstream homology arm-downstream is homologous the upstream and downstream homology arm that need to knock out gene
Arm)；Recombinant fragment for integration forms that (upstream is homologous with the upstream and downstream homology arm and genetic fragment to be integrated of integration site
Arm-target gene-downstream homology arm).Using primer-design software primer5, with gene to be knocked out or the upper of integration site is waited for
Downstream sequence is template, design upstream and downstream homology arm primer (amplification length about 400-500bp)；To wait for integrator gene as template,
The amplimer of design integration gene.After expanding upstream and downstream homology arm and target gene fragment respectively by the method for PCR, then pass through
Lap over PCR Prepare restructuring segments.Archaeal dna polymerase used in PCR is purchased from TaKaRa companies, includes the PrimeSTAR HS of high-fidelity
DNA Polymerase and PCR product carry the system and method for Ex.taq the DNA Polymerase, PCR of cohesive end
Such as following table：
HS enzyme PCR amplification systems
Ex.taq enzyme PCR amplification systems
The system of over-lap PCR such as following table：
HS enzyme over-lap PCR amplification systems
Note：Template is made of the amplified fragments and target gene equimolar of upstream and downstream homology arm, and total amount is no more than 10ng.
Ex.taq enzyme over-lap PCR amplification systems
Note：Template is made of the amplified fragments and target gene equimolar of upstream and downstream homology arm, and total amount is no more than 10ng.
PCR reaction conditions：Pre-degeneration (95 DEG C) 5min；Then 30 wheel cycles are carried out：It is denaturalized (98 DEG C) 10s, anneal ((Tm-
3/5) DEG C) 15s, 72 DEG C extend (this enzyme activity 1min extends about 1kb)；72 DEG C are continued to extend 10min；It maintains (4 DEG C).
The conversion of 1.3 plasmids and recombinant dna fragment
1.3.1pREDCas9 conversion
The electricity that pREDCas9 plasmid electricity is gone to W3110 using the method that electricity turns is turned in competence, and thalline is recovered and is cultivated
It is coated on afterwards on the LB tablets containing miramycin, 32 DEG C are incubated overnight.Single bacterium colony is grown in resistant panel carries out bacterium with identification primer
PCR is fallen, positive recombinant is screened.
1.3.2 prepared by the purpose bacterial strain electrotransformation competence containing pREDCas9
32 DEG C are cultivated to OD600When=0.1~0.2, the IPTG (making its final concentration of 0.1mM) of 0.1M is added, continues to cultivate
To OD600Competence preparation is carried out when=0.6~0.7.The purpose for adding IPTG is the recombination enzyme induction made on pREDCas9 plasmids
Expression.Competence prepares required culture medium and preparation process is operated with reference to conventional criteria.
1.3.3pGRB with the conversion of recombinant dna fragment
By pGRB and donor DNA segment, electrotransformation to the electricity containing pREDCas9 turns in competent cell simultaneously.Electricity is turned
The thalline of culture of recovering after change is coated on the LB tablets containing ampicillin and miramycin, and 32 DEG C are incubated overnight.It is same with upstream
The downstream primer of source arm sense primer and downstream homology arm, or special identification primer is designed, carry out bacterium colony PCR verifications, screening
Positive recombinant simultaneously protects bacterium.
The elimination of 1.4 plasmids
1.4.1 the elimination of pGRB
Positive recombinant is placed in the LB culture mediums containing 0.2% arabinose and is incubated overnight, is coated with after appropriate dilution
In on the LB tablets containing miramycin resistance, 32 DEG C are incubated overnight.It is flat to LB of the point containing ampicillin and miramycin resistance
Plate is selected ampicillin plate and is not grown, and the single bacterium colony of miramycin resistant panel growth protects bacterium.
1.4.2 the elimination of pREDCas9 plasmids
Positive recombinant is transferred in the LB liquid medium of non-resistant, 42 DEG C are incubated overnight, and are coated with after appropriate dilution
In on the LB tablets of non-resistant, 37 DEG C are incubated overnight.To putting the LB tablets containing miramycin resistance and non-resistant, miramycin is selected
Resistant panel is not grown, and the single bacterium colony of non-resistant plated growth protects bacterium.
The genetic engineering of 2.L- tryptophan engineered strain E.coli TRP03 combines transformation process
TrpE (S40F) is introduced while 2.1 tryptophan operon promoters are replaced to be mutated
2.1.1 the carrier T of structure mutation containing trpE (S40F)
Using E.coli W3110 (ATCC27325) genomes as template, according to trpE genes and its upstream and downstream sequence design
TrpE (S40F) is mutated upstream homology arm primer (trpE (S40F) -1, trpE (S40F) -2) and trpE (S40F) is mutated downstream
Homology arm primer (trpE (S40F) -3, trpE (S40F) -4), wherein trpE (S40F) -2, trpE (S40F) -3 is simultaneously containing mutation
Its upstream and downstream homology arm segment of Ex.taq enzymes PCR amplification is used in combination in sequence (TCC sports TTT) and reverse complemental.Above-mentioned segment is logical
The method for crossing Ex.taq enzyme over-lap PCRs obtains the segment (upstream homology arm-downstream homology arm) of the mutation containing trpE (S40F).By institute
Obtain segment and carrier T in molar ratio 4:1 mixing (total amount is no more than 100ng), is added the solution I ligases of 5 μ L, adds water
It mends to 10 μ L systems, after 16 DEG C connect 1h, selects positive transformant by the operation of method shown in 1.1.5, extraction plasmid send sequencing true
Fixed successfully structure T loads-trpE (S40F).
2.1.2 tryptophan operon is transformed
Using E.coli W3110 (ATCC27325) genomes as template, it is same that upstream is designed according to trpE gene upstream sequences
Source arm primer (trpE (S40F) -1, trpE (S40F) -5)；It is same according to sequence design downstream with T loads-trpE (S40F) for template
Source arm primer (trpE (S40F) -6, trpE (S40F) -4), wherein the design addition of trc promoter sequences is in primer trpE
(S40F) on -5 and trpE (S40F) -6, with its upstream and downstream homology arm segment of HS enzymes PCR amplification.Above-mentioned segment is overlapped by HS enzymes
(upstream homology arm-Ptrc trpE (S40F)-downstream is same for the replacement segment of method acquisition Ptrc trpE (S40F) gene of PCR
Source arm).Building pGRB-trpL, (gene trpL is located at tryptophan operon promoter upstream, and codes for amino acid tryptophan operon is leading
Peptide) DNA fragmentation containing target sequence that uses is made by the annealing of primer pGRB-trpL-F and pGRB-trpL-R.It prepares
The competent cell of E.coli W3110 is operated according to method shown in 1.3 and 1.4, final to obtain bacterial strain E.coli TRP01.
The electrophoretogram that Ptrc trpE (S40F) gene replaces the PCR verifications of the structure and positive strain of segment is shown in attached drawing 2.Wherein, upstream
The length of homology arm should be 620bp, and the length of downstream homology arm (including trpE (S40F)) should be 875bp, Ptrc trpE
(S40F) overall length of gene replacement segment should be 1570bp, when PCR is verified, identification primer (trpE (S40F) -7) is designed and is existed
Inside Ptrc promoter sequences, positive bacteria pcr amplified fragment length should be 667bp, and opportunistic pathogen PCR should be without band.
2.2 Ptrc aroG (S180F) gene integrations are to the sites genome tyrR
2.2.1 the carrier T of structure mutation containing aroG (S180F)
Using E.coli W3110 (ATCC27325) genomes as template, according to aroG genes and its upstream and downstream sequence design
AroG (S180F) is mutated upstream homology arm primer (aroG (S180F) -1, aroG (S180F) -2) and aroG (S180F) mutation
Downstream homology arm primer (aroG (S180F) -3, aroG (S180F) -4), wherein aroG (S180F) -2, aroG (S180F) -3 are same
When contain mutant nucleotide sequence (TCT sports TTT) and reverse complemental, its upstream and downstream homology arm segment of Ex.taq enzymes PCR amplification is used in combination.On
Segment is stated by the methods of Ex.taq enzyme over-lap PCRs to obtain the segment of the mutation containing aroG (S180F) (upstream homology arm-downstream is same
Source arm).By obtained segment and carrier T in molar ratio 4:1 mixing (total amount is no more than 100ng), the solution I that 5 μ L are added connect
Enzyme is connect, water is added to mend to 10 μ L systems, after 16 DEG C connect 1h, positive transformant is selected by the operation of method shown in 1.1.5, extracts plasmid
Sequencing is sent to determine successfully structure T loads-aroG (S180F).
2.2.2 the integration of Ptrc aroG (S180F) gene
Using E.coli W3110 (ATCC27325) genomes as template, according in the upstream and downstream sequence design of tyrR genes
Homology arm primer (tyrR-1, tyrR-2) and downstream homology arm primer (tyrR-3, tyrR-4) are swum, with the T loads-aroG of structure
(S180F) it is template, according to the primers (aroG (S180F) -5, aroG (S180F) -6, wherein trc of aroG genes
Promoter sequence design addition is on primer tyrR-2 and aroG (S180F) -5, with HS enzyme PCR amplification Ptrc aroG (S180F)
Genetic fragment.Above-mentioned segment obtains the integration segment (upstream of Ptrc aroG (S180F) gene by the method for HS enzyme over-lap PCRs
Homology arm-Ptrc aroG (S180F)-downstream homology arm).The DNA fragmentation containing target sequence that structure pGRB-tyrR is used passes through
The annealing of primer pGRB-tyrR-F and pGRB-tyrR-R are made.The competent cell for preparing E.coli TRP01, according to 1.3 Hes
Method shown in 1.4 operates, final to obtain bacterial strain E.coli TRP02.The structure of Ptrc aroG (S180F) gene integration segment
See attached drawing 3 with the PCR of the positive strain electrophoretograms verified.Wherein, the length of upstream homology arm should be 347bp, downstream homology arm
Length should be 507bp, the length of Ptrc aroG (S180F) genetic fragment should be 1285bp, Ptrc aroG (S180F) gene
The whole length for integrating segment should be 2139bp, and when PCR is verified, positive bacteria pcr amplified fragment length should be 2139bp, opportunistic pathogen
Pcr amplified fragment length should be 1493bp.
2.3 Plac serA (H344A, N364A) gene integrations are to the sites genome yjiV
2.3.1 the carrier T of structure mutation containing serA (H344A, N364A)
Using E.coli W3110 (ATCC27325) genomes as template, according to serA genes and its upstream and downstream sequence design
SerA (H344A) is mutated under upstream homology arm primer (serA (H344A) -1, serA (H344A) -2) and serA (H344A) mutation
Homology arm primer (serA (H344A) -3, serA (H344A) -4) is swum, wherein serA (H344A) -2, serA (H344A) -3 is simultaneously
Containing mutant nucleotide sequence (CAC sports GCC) and reverse complemental, its upstream and downstream homology arm segment of Ex.taq enzymes PCR amplification is used in combination.It is above-mentioned
Segment by the methods of Ex.taq enzyme over-lap PCRs obtains the segment of the mutation containing serA (H344A), and (upstream homology arm-downstream is homologous
Arm).By obtained segment and carrier T in molar ratio 4:1 mixing (total amount is no more than 100ng), is added the solution I connections of 5 μ L
Enzyme adds water to mend to 10 μ L systems, after 16 DEG C connect 1h, selects positive transformant by the operation of method shown in 1.1.5, extraction plasmid is sent
Sequencing determines successfully structure T loads-serA (H344A).
With T loads-serA (H344A) for template, it is mutated according to serA genes and its upstream and downstream sequence design serA (N364A)
Upstream homology arm primer (serA (H344A) -1, serA (N364A) -1) and serA (N364A) are mutated downstream homology arm primer
(serA (N364A) -2, serA (H344A) -4), wherein serA (N364A) -1, serA (N364A) -2 contain mutant nucleotide sequence simultaneously
Its upstream and downstream homology arm segment of Ex.taq enzymes PCR amplification is used in combination in (AAC sports GCC) and reverse complemental.Above-mentioned segment passes through
(upstream homology arm-downstream is homologous for the segment of method acquisition mutation containing serA (H344A, N364A) of Ex.taq enzyme over-lap PCRs
Arm).By obtained segment and carrier T in molar ratio 4:1 mixing (total amount is no more than 100ng), is added the solution I connections of 5 μ L
Enzyme adds water to mend to 10 μ L systems, after 16 DEG C connect 1h, selects positive transformant by the operation of method shown in 1.1.5, extraction plasmid is sent
Sequencing determines successfully structure T loads-serA (H344A, N364A).
2.3.2 the integration of Plac serA (H344A, N364A) gene
Using E.coli W3110 (ATCC27325) genomes as template, according in the upstream and downstream sequence design of yjiV genes
Swim homology arm primer (yjiV-1, yjiV-2) and downstream homology arm primer (yjiV-3, yjiV-4)；With T loads-serA (H344A,
N364A it is) template, according to its primers (serA (H344A, N364A) -1, serA (H344A, N364A) -2),
In, the design addition of lac promoter sequences is on primer yjiV-2 and serA (H344A, N364A) -1, with HS enzyme PCR amplifications Plac
SerA (H344A, N364A) genetic fragment.Above-mentioned segment by the methods of HS enzyme over-lap PCRs obtain Plac serA (H344A,
N364A) the integration segment (upstream homology arm-Plac serA (H344A, N364A)-downstream homology arm) of gene.Build pGRB-
The DNA fragmentation containing target sequence that yjiV is used is made by the annealing of primer pGRB-yjiV-F and pGRB-yjiV-R.It prepares
The competent cell of E.coli TRP02 is operated according to method shown in 1.3 and 1.4, final to obtain bacterial strain E.coli TRP03.
The electrophoretogram of the PCR verifications of the structure and positive strain of Plac serA (H344A, N364A) gene integration segment is shown in attached drawing 4.Its
In, the length of upstream homology arm should be 505bp, and the length of downstream homology arm should be 581bp, Plac serA (H344A, N364A)
The length of genetic fragment should be 1379bp, and whole length of Plac serA (H344A, N364A) gene integration segment should be
When 2503bp, PCR are verified, positive bacteria pcr amplified fragment length should be 2503bp, and opportunistic pathogen pcr amplified fragment length should be
2252bp。
3. involved primer see the table below during strain improvement：
Embodiment 2：Introduce the glutamine synthelase from lactobacillus acidophilus (Lactobacillus acidophilus)
(glnA) is to L-Trp engineering bacteria
1.glnA gene chemical synthesis
According to lactobacillus acidophilus (Lactobacillus acidophilus) the paddy ammonia announced on the GENBANK in NCBI
Amide synthetase coding gene sequence, carrying out codon optimization to it with common codon software in Escherichia coli, (codon is excellent
Change front and back sequence respectively such as SEQ ID NO：8 and SEQ ID NO：Shown in 9) so that it can the efficient transcription in Escherichia coli.
Sequence after optimization is sent to Jin Weizhi companies to synthesize, obtains the recombinant plasmid pUC57glnA with glnA genes, wherein enzyme
Enzyme site is Hind III and BamH I, is stored in Escherichia coli.
2.Plac glnA are integrated into genome ycjV pseudogenes site
Using E.coli W3110 (ATCC27325) genomes as template, according to the upstream and downstream sequence design of ycjV pseudogenes
Upstream homology arm primer (ycjV-1, ycjV-2), downstream homology arm primer (ycjV-3, ycjV-4), with recombinant plasmid
PUC57glnA is template, according to its sequence design Plac glnA fragment primers (glnA-1, glnA-2), HS enzymes PCR is used in combination to expand
Increase its upstream and downstream homology arm segment and Plac glnA segments.Above-mentioned segment obtains Plac by the method for HS enzyme over-lap PCRs
The integration segment (the upstream downstreams homology arm-Plac glnA- homology arm) of glnA genes.The sequence containing target that structure pGRB-ycjV is used
The DNA fragmentation of row is made by the annealing of primer pGRB-ycjV-F and pGRB-ycjV-R.Prepare the impression of E.coli TRP03
State cell is operated according to method shown in 1.3 and 1.4, final to obtain bacterial strain E.coli TRP04.Plac glnA gene integrations
The electrophoretogram of the PCR verifications of the structure and positive strain of segment is shown in attached drawing 5.Wherein, the length of upstream homology arm should be 452bp,
The length of downstream homology arm should be 558bp, and the length of Ptrc glnA genetic fragments should be 1414bp, Plac glnA gene integrations
Whole length of segment should be 2424bp, and when PCR is verified, positive bacteria pcr amplified fragment length should be 2424bp, and opportunistic pathogen PCR expands
Increase fragment length and should be 1859bp.
Embodiment 3：L-Trp is produced using Recombinant organism shake flask fermentation
A kind of concrete operations that shake flask fermentation production L-Trp is carried out using Recombinant organism are as follows：
Inclined-plane culture：It takes -80 DEG C of preservation of bacteria strain streak inoculations in activated inclined plane, 37 DEG C of culture 12h, and passes on primary；
Shake-flask seed culture：A ring inclined-plane seed, which is scraped, with oese is inoculated in the 500mL equipped with 30mL seed culture mediums
In triangular flask, nine layers of gauze sealing, 37 DEG C, 200rpm cultivates 8-10h；
Shake flask fermentation culture：It is inoculated into the 500mL triangular flasks equipped with fermentation medium by 10-15% (v/v) inoculum concentration
(final volume 30mL), nine layers of gauze seal, 37 DEG C, 200r/min shaken cultivations, are maintained in fermentation process by adding ammonium hydroxide
PH is in 7.0-7.2；Adding 60% (m/v) glucose solution maintains fermentation (give instruction agent with phenol red, zymotic fluid color is not
It is considered as when changing again and lacks sugar, adds 1-2mL 60% (m/v) glucose solution when lacking sugar, make the concentration of glucose in zymotic fluid
For initial value 20-40g/L)；Fermentation period 22-26h；
L-Trp concentration mensuration in zymotic fluid：It collects zymotic fluid 1mL, 13000rpm and centrifuges 1min, collect supernatant.Make
It is diluted to certain multiple (being diluted to 0.1-0.5g/L) by supernatant is collected with deionized water, liquid is used after 0.22 μm of micro porous filtration
Mutually detection L-Trp content, chromatographic condition are：Chromatographic column Kromasil C18 columns (250mm × 460mm, 5 μm), mobile phase
For 10% acetonitrile solution, flow velocity 1.0mL/min, 40 DEG C, Detection wavelength 278nm of column temperature, sample size is 20 μ L, and appearance time is about
The concentration of L-Trp in zymotic fluid is calculated according to its peak area by the standard curve of drafting by 3.752min；
The drafting of L-Trp standard curve：L-Trp a concentration of 0.1g/L, 0.2g/L, 0.3g/L, 0.4g/ are taken respectively
The solution of L, 0.5g/L obtain the corresponding peak area of L-Trp of respective concentration according to above-mentioned chromatographic condition with liquid phase measurement,
Drafting obtains L-Trp concentration and the relevant criterion curve of its peak area is as shown in Figure 6.
Activated inclined plane nutrient media components are：Glucose 1-3g/L, tryptone 5-10g/L, beef extract 5-10g/L, yeast
Extract 2-5g/L, NaCl 2-5g/L, agar 15-30g/L, remaining is water, pH 7.0-7.2,121 DEG C of high steam pot sterilizings
20min；
Seed culture medium group is divided into：Glucose 20-40g/L, (NH4)2SO41-5g/L, KH2PO41-5g/L, MgSO4·
7H2O 0.5-2g/L, yeast extract 2-5g/L, FeSO4·7H2O 1-3mg/L, MnSO4·H2O 1-3mg/L, VH 0.1-
0.5mg/L, VB10.5-1.0mg/L, micro-mixed liquor 1-3ml/L, phenol red 15-30g/L, remaining is water, pH 7.0-
7.2,115 DEG C of high steam pots sterilizing 15min；
Fermentation medium group is divided into：Glucose 20-40g/L, (NH4)2SO42-6g/L, KH2PO41-5g/L, MgSO4·
7H2O 0.5-2g/L, yeast extract 1-5g/L, FeSO4·7H2O 30-60mg/L, MnSO4·7H2O 1-5mg/L, VH
0.1-0.5mg/L, VB10.5-1.0mg/L, micro-mixed liquor 1-3ml/L, phenol red 15-30g/L, remaining is water, pH
7.0-7.2,115 DEG C of high steam pots sterilizing 15min；
Micro-mixed liquor group is divided into：Na2MoO4·2H2O 2.5g/L, AlCl3·6H2O 2.5g/L, NiSO4·
6H2O 2.5g/L, CoCl2·6H2O 1.75g/L, CaCl2·2H2O 10g/L, ZnSO4·7H2O 0.5g/L, CuCl2·2H2O
0.25g/L, H3BO3 0.125g/L。
Shake flask fermentation is carried out using L-Trp engineered strain E.coli TRP03, the E.coli TRP04 of above-mentioned structure,
With the L-Trp work for the carrying plasmid that starting strain E.coli W3110 and University Of Science and Technology Of Tianjin's metabolic engineering laboratory preserve
Journey bacterial strain E.coli TRTH (bibliography Qian L, Cheng Y, Xie X, et al.Modification of
tryptophan transport system and its impact on production of L-tryptophan in
Escherichia coli[J].Bioresource Technology,2012,114(2):549.) equal conditions fermentation conduct
Control, the results are shown in Figure 7 for experiment.Starting strain E.coli W3110 hardly accumulate L-Trp, engineered to combine
The L-Trp engineered strain E.coli TRP03 arrived accumulate L-Trp up to 8.75 ± 0.75g/L, have not only reached accumulation L-
The purpose of tryptophan, have also exceeded carry the L-Trp engineered strain E.coli TRTH accumulation L-Trp 6.75 of plasmid ±
The yield of 0.84g/L, and introduce the L-Trp engineered strain E.coli TRP04 products after glutamine synthetase gene glnA
Tired L-Trp improves 28.57% on the basis of E.coli TRP03 again, up to 11.55 ± 0.55g/L.
Embodiment 4：L-Trp is produced using Recombinant organism 5L ferment tanks
1. a kind of concrete operations for normally producing L-Trp using Escherichia coli progress 5L fermentation tanks are as follows：
Slant activation culture：It is protected in tube from -80 DEG C of refrigerators and scrapes a ring strain, be spread evenly across activated inclined plane, 37 DEG C of trainings
12-16h is supported, switching eggplant-shape bottle continues to cultivate 12-16h；
Seed culture：It takes appropriate amounts of sterilized water in eggplant-shape bottle, bacteria suspension is accessed in seed culture medium, pH stablizes 7.0
Left and right, temperature is constant at 37 DEG C, and for dissolved oxygen between 25-35%, culture to dry cell weight reaches 5-6g/L；
Fermented and cultured：Fresh fermentation medium is accessed according to 15-20% inoculum concentrations, is started to ferment, be controlled in fermentation process
PH processed stablizes 7.0 or so, and temperature maintains 37 DEG C, and dissolved oxygen is between 25-35%；When the glucose consumption in culture medium it is complete it
Afterwards, the glucose solution of stream plus 80% (m/v) maintains the concentration of glucose in fermentation medium in 0.1-5g/L；Fermentation period
35-42h；
Activated inclined plane nutrient media components are：Glucose 1-3g/L, tryptone 5-10g/L, beef extract 5-10g/L, yeast
Extract 2-5g/L, NaCl 2-5g/L, agar 15-30g/L, remaining is water, pH 7.0-7.2,121 DEG C of high steam pot sterilizings
20min；
Seed culture medium group is divided into：Glucose 20-40g/L, (NH4)2SO41-5g/L, KH2PO41-5g/L, MgSO4·
7H2O 0.5-2g/L, yeast extract 2-5g/L, FeSO4·7H2O 1-3mg/L, MnSO4·H2O 1-3mg/L, VH 0.1-
0.5mg/L, VB10.5-1.0mg/L, micro-mixed liquor 1-3ml/L, remaining is water, pH 7.0-7.2,115 DEG C of high pressures steamings
Steam-boiler sterilizing 15min；
Fermentation medium group is divided into：Glucose 20-40g/L, (NH4)2SO42-6g/L, KH2PO41-5g/L, MgSO4·
7H2O 0.5-2g/L, yeast extract 1-5g/L, FeSO4·7H2O 30-60mg/L, MnSO4·7H2O 1-5mg/L, VH
0.1-0.5mg/L, VB10.5-1.0mg/L, micro-mixed liquor 1-3ml/L, remaining is water, pH 7.0-7.2,115 DEG C of height
Press steam copper sterilizing 15min；
Micro-mixed liquor group is divided into：Na2MoO4·2H2O 2.5g/L, AlCl3·6H2O 2.5g/L, NiSO4·
6H2O 2.5g/L, CoCl2·6H2O 1.75g/L, CaCl2·2H2O 10g/L, ZnSO4·7H2O 0.5g/L, CuCl2·2H2O
0.25g/L, H3BO3 0.125g/L。
5L fermentation tank shaking flasks are carried out using the best L-Trp engineered strain E.coli TRP04 of shake flask fermentation result to send out
Ferment, the L-Trp engineered strain E.coli TRTH equal conditions to carry plasmid ferment as a contrast, and experimental result is as schemed
Shown in 8.The concentration of L-Trp in the method monitoring fermentation tank of L-Trp concentration in zymotic fluid is detected by above-mentioned shake flask fermentation,
E.coli TRP04 fermentations 40h can accumulate L-Trp and reach 44.1g/L, and 15.1% is improved compared to E.coli TRTH.
Although above-mentioned have been disclosed the preferable embodiment of the present invention, it is not limited to the present invention, any ability
Field technique personnel can be various change and modification, therefore the guarantor of the present invention without departing from the spirit and scope of the present invention
Shield range should be subject to defined by claims.
Sequence table
<110>University Of Science and Technology Of Tianjin
<120>The structure of Recombinant organism and its purposes of production L-Trp
<141> 2018-06-29
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 74
<212> DNA
<213> E. coli W3110
<400> 1
ttgacaatta atcatccggc tcgtataatg tgtggaattg tgagcggata acaatttcac 60
acaggaaaca gacc 74
<210> 2
<211> 1563
<212> DNA
<213> E. coli W3110
<400> 2
atgcaaacac aaaaaccgac tctcgaactg ctaacctgcg aaggcgctta tcgcgacaat 60
cccaccgcgc tttttcacca gttgtgtggg gatcgtccgg caacgctgct gctggaattt 120
gcagatatcg acagcaaaga tgatttaaaa agcctgctgc tggtagacag tgcgctgcgc 180
attacagctt taggtgacac tgtcacaatc caggcacttt ccggcaacgg cgaagccctc 240
ctggcactac tggataacgc cctgcctgcg ggtgtggaaa gtgaacaatc accaaactgc 300
cgtgtgctgc gcttcccccc tgtcagtcca ctgctggatg aagacgcccg cttatgctcc 360
ctttcggttt ttgacgcttt ccgtttattg cagaatctgt tgaatgtacc gaaggaagaa 420
cgagaagcca tgttcttcgg cggcctgttc tcttatgacc ttgtggcggg atttgaagat 480
ttaccgcaac tgtcagcgga aaataactgc cctgatttct gtttttatct cgctgaaacg 540
ctgatggtga ttgaccatca gaaaaaaagc acccgtattc aggccagcct gtttgctccg 600
aatgaagaag aaaaacaacg tctcactgct cgcctgaacg aactacgtca gcaactgacc 660
gaagccgcgc cgccgctgcc agtggtttcc gtgccgcata tgcgttgtga atgtaatcag 720
agcgatgaag agttcggtgg cgtagtgcgt ttgttgcaaa aagcgattcg cgctggagaa 780
attttccagg tggtgccatc tcgccgtttc tctctgccct gcccgtcacc gctggcggcc 840
tattacgtgc tgaaaaagag taatcccagc ccgtacatgt tttttatgca ggataatgat 900
ttcaccctat ttggcgcgtc gccggaaagc tcgctcaagt atgatgccac cagccgccag 960
attgagatct acccgattgc cggaacacgc ccacgcggtc gtcgcgccga tggttcactg 1020
gacagagatc tcgacagccg tattgaactg gaaatgcgta ccgatcataa agagctgtct 1080
gaacatctga tgctggttga tctcgcccgt aatgatctgg cacgcatttg cacccccggc 1140
agccgctacg tcgccgatct caccaaagtt gaccgttatt cctatgtgat gcacctcgtc 1200
tctcgcgtag tcggcgaact gcgtcacgat cttgacgccc tgcacgctta tcgcgcctgt 1260
atgaatatgg ggacgttaag cggtgcgccg aaagtacgcg ctatgcagtt aattgccgag 1320
gcggaaggtc gtcgccgcgg cagctacggc ggcgcggtag gttatttcac cgcgcatggc 1380
gatctcgaca cctgcattgt gatccgctcg gcgctggtgg aaaacggtat cgccaccgtg 1440
caagcgggtg ctggtgtagt ccttgattct gttccgcagt cggaagccga cgaaacccgt 1500
aacaaagccc gcgctgtact gcgcgctatt gccaccgcgc atcatgcaca ggagactttc 1560
tga 1563
<210> 3
<211> 1053
<212> DNA
<213> E. coli W3110
<400> 3
atgaattatc agaacgacga tttacgcatc aaagaaatca aagagttact tcctcctgtc 60
gcattgctgg aaaaattccc cgctactgaa aatgccgcga atacggttgc ccatgcccga 120
aaagcgatcc ataagatcct gaaaggtaat gatgatcgcc tgttggttgt gattggccca 180
tgctcaattc atgatcctgt cgcggcaaaa gagtatgcca ctcgcttgct ggcgctgcgt 240
gaagagctga aagatgagct ggaaatcgta atgcgcgtct attttgaaaa gccgcgtacc 300
acggtgggct ggaaagggct gattaacgat ccgcatatgg ataatagctt ccagatcaac 360
gacggtctgc gtatagcccg taaattgctg cttgatatta acgacagcgg tctgccagcg 420
gcaggtgagt ttctcgatat gatcacccca caatatctcg ctgacctgat gagctggggc 480
gcaattggcg cacgtaccac cgaatcgcag gtgcaccgcg aactggcatc agggcttttt 540
tgtccggtcg gcttcaaaaa tggcaccgac ggtacgatta aagtggctat cgatgccatt 600
aatgccgccg gtgcgccgca ctgcttcctg ttcgtaacga aatgggggca ttcggcgatt 660
gtgaatacca gcggtaacgg cgattgccat atcattctgc gcggcggtaa agagcctaac 720
tacagcgcga agcacgttgc tgaagtgaaa gaagggctga acaaagcagg cctgccagca 780
caggtgatga tcgatttcag ccatgctaac tcgtccaaac aattcaaaaa gcagatggat 840
gtttgtgctg acgtttgcca gcagattgcc ggtggcgaaa aggccattat tggcgtgatg 900
gtggaaagcc atctggtgga aggcaatcag agcctcgaga gcggggagcc gctggcctac 960
ggtaagagca tcaccgatgc ctgcatcggc tgggaagata ccgatgctct gttacgtcaa 1020
ctggcgaatg cagtaaaagc gcgtcgcggg taa 1053
<210> 4
<211> 74
<212> DNA
<213> E. coli W3110
<400> 4
tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca 60
cacaggaaac agct 74
<210> 5
<211> 1233
<212> DNA
<213> E. coli W3110
<400> 5
atggcaaagg tatcgctgga gaaagacaag attaagtttc tgctggtaga aggcgtgcac 60
caaaaggcgc tggaaagcct tcgtgcagct ggttacacca acatcgaatt tcacaaaggc 120
gcgctggatg atgaacaatt aaaagaatcc atccgcgatg cccacttcat cggcctgcga 180
tcccgtaccc atctgactga agacgtgatc aacgccgcag aaaaactggt cgctattggc 240
tgtttctgta tcggaacaaa ccaggttgat ctggatgcgg cggcaaagcg cgggatcccg 300
gtatttaacg caccgttctc aaatacgcgc tctgttgcgg agctggtgat tggcgaactg 360
ctgctgctat tgcgcggcgt gccggaagcc aatgctaaag cgcaccgtgg cgtgtggaac 420
aaactggcgg cgggttcttt tgaagcgcgc ggcaaaaagc tgggtatcat cggctacggt 480
catattggta cgcaattggg cattctggct gaatcgctgg gaatgtatgt ttacttttat 540
gatattgaaa ataaactgcc gctgggcaac gccactcagg tacagcatct ttctgacctg 600
ctgaatatga gcgatgtggt gagtctgcat gtaccagaga atccgtccac caaaaatatg 660
atgggcgcga aagaaatttc actaatgaag cccggctcgc tgctgattaa tgcttcgcgc 720
ggtactgtgg tggatattcc ggcgctgtgt gatgcgctgg cgagcaaaca tctggcgggg 780
gcggcaatcg acgtattccc gacggaaccg gcgaccaata gcgatccatt tacctctccg 840
ctgtgtgaat tcgacaacgt ccttctgacg ccacacattg gcggttcgac tcaggaagcg 900
caggagaata tcggcctgga agttgcgggt aaattgatca agtattctga caatggctca 960
acgctctctg cggtgaactt cccggaagtc tcgctgccac tgcacggtgg gcgtcgtctg 1020
atgcacatcg ccgaaaaccg tccgggcgtg ctaactgcgc tgaacaaaat cttcgccgag 1080
cagggcgtcg ccatcgccgc gcaatatctg caaacttccg cccagatggg ttatgtggtt 1140
attgatattg aagccgacga agacgttgcc gaaaaagcgc tgcaggcaat gaaagctatt 1200
ccgggtacca ttcgcgcccg tctgctgtac taa 1233
<210> 6
<211> 1542
<212> DNA
<213> E. coli W3110
<400> 6
atgcgtctgg aagtcttttg tgaagaccga ctcggtctga cccgcgaatt actcgatcta 60
ctcgtgctaa gaggcattga tttacgcggt attgagattg atcccattgg gcgaatctac 120
ctcaattttg ctgaactgga gtttgagagt ttcagcagtc tgatggccga aatacgccgt 180
attgcgggtg ttaccgatgt gcgtactgtc ccgtggatgc cttccgaacg tgagcatctg 240
gcgttgagcg cgttactgga ggcgttgcct gaacctgtgc tctctgtcga tatgaaaagc 300
aaagtggata tggcgaaccc ggcgagctgt cagctttttg ggcaaaaatt ggatcgcctg 360
cgcaaccata ccgccgcaca attgattaac ggctttaatt ttttacgttg gctggaaagc 420
gaaccgcaag attcgcataa cgagcatgtc gttattaatg ggcagaattt cctgatggag 480
attacgcctg tttatcttca ggatgaaaat gatcaacacg tcctgaccgg tgcggtggtg 540
atgttgcgat caacgattcg tatgggccgc cagttgcaaa atgtcgccgc ccaggacgtc 600
agcgccttca gtcaaattgt cgccgtcagc ccgaaaatga agcatgttgt cgaacaggcg 660
cagaaactgg cgatgctaag cgcgccgctg ctgattacgg gtgacacagg tacaggtaaa 720
gatctctttg cctacgcctg ccatcaggca agccccagag cgggcaaacc ttacctggcg 780
ctgaactgtg cgtctatacc ggaagatgcg gtcgagagtg aactgtttgg tcatgctccg 840
gaagggaaga aaggattctt tgagcaggcg aacggtggtt cggtgctgtt ggatgaaata 900
ggggaaatgt caccacggat gcaggcgaaa ttactgcgtt tccttaatga tggcactttc 960
cgtcgggttg gcgaagacca tgaggtgcat gtcgatgtgc gggtgatttg cgctacgcag 1020
aagaatctgg tcgaactggt gcaaaaaggc atgttccgtg aagatctcta ttatcgtctg 1080
aacgtgttga cgctcaatct gccgccgcta cgtgactgtc cgcaggacat catgccgtta 1140
actgagctgt tcgtcgcccg ctttgccgac gagcagggcg tgccgcgtcc gaaactggcc 1200
gctgacctga atactgtact tacgcgttat gcgtggccgg gaaatgtgcg gcagttaaag 1260
aacgctatct atcgcgcact gacacaactg gacggttatg agctgcgtcc acaggatatt 1320
ttgttgccgg attatgacgc cgcaacggta gccgtgggcg aagatgcgat ggaaggttcg 1380
ctggacgaaa tcaccagccg ttttgaacgc tcggtattaa cccagcttta tcgcaattat 1440
cccagcacgc gcaaactggc aaaacgtctc ggcgtttcac ataccgcgat tgccaataag 1500
ttgcgggaat atggtctgag tcagaagaag aacgaagagt aa 1542
<210> 7
<211> 1606
<212> DNA
<213> E. coli W3110
<400> 7
gtgtatgtga accagcgaca aaattgtgac tgtggaagcc ctgtatacga agttgcattc 60
tgtaatgatt gtaatgagcc tcatcttctg gcacgggaca aaaagggcaa actagtccag 120
tgggaaaata aaggtggcga tgaattctct ttgcaggatg aagtacctgt tgaacatgac 180
gctacagaag aaaaagtcga aaaagagaac agttttcagc ctccgttgat tattgccgca 240
ggagagacca gcgaggcagg ttatacccta caacgcctcg accgtcagac gcgccgtatt 300
ggcgttatta acaatgacag cattccgctg attattaatg atattgaaca ggtttgtagt 360
gccagtggct gtggctacag aggcatgagt gggaaacagc ccttccggcg tgcactatta 420
ggtgggccat tttacgttac taatatcgtg cccaccgttc tagagtattg tcaggacttt 480
accagtgatg aaggcaaaga gggcgtcgga ccagattcgt tgccaggacg aggtcgtcga 540
ctcatcacct ttacagacag tcgacaaggg acagcccgaa tggcggtgcg tatgcagcaa 600
gaagcagaac gcagtcgctt acgcggaagc gtagtcgaaa ttctcagctg gcatcaaagg 660
acgcaaacgt ctacagcgcc gaatgccaat gccgatctgg aaaaattagc ggccagggcg 720
aagcaagccc gtgagcaggc agaagaatat cgaagctggg gaatgccaga ccaggcgaaa 780
ttgtcacaag cacaggctga acagcttgaa caggcttatc aggctgcaac cggtgggaaa 840
gccgcgacta tcctggtatc ccgaacctgg acggagatgg ttaacgagct taaagagaga 900
gccgatatcc gcgggccggt tctgcaatat aaccattatc ttaagcctga agtgtttaat 960
gaaaacggcg gcccccttaa gctttctgaa atgttgttgt tccgggaatt catgcgtcgc 1020
cctaaacgga ctaacagcct ggaaacacag gggctggttc aggttggtta ccaggggctg 1080
gagaaaatac ataagagccc cttgcactgg caggaaaaag gattaacgct ggatgactgg 1140
cgcgattttc tcaaggttac gttggatcat tacgttcgcg agagcaactt cacacagctg 1200
gatgatgagc tgaaaaactg gattggtagc cgtttttcat caaaattcgt ccgtaacccg 1260
gaatcaaaag atcctgaaga taatcagaac agacgctggc ctcaaattcg taatggcaac 1320
gtatcccatc gtttagcgaa gttgctgatg ctgggggctg gattcaaaac cgtcaatgcg 1380
gcaactattg atattatcaa tacatggctg aaagaagcat gggcccaact taccggaccg 1440
cttgcagtac tgaaacccga tggcaaccgt ttttatttac cgaaagagca tatgactttt 1500
tctttaatca cggatgcctg gatttgcccg gtaaccaata aaatcctgga tacggctttt 1560
aaaggcttaa ccccttatct gcctacccat atttcgttcg agcatc 1606
<210> 8
<211> 1338
<212> DNA
<213> Lactobacillus acidophilus
<400> 8
atgagtaaac aatacactgc agaagaaatt aaaacagaag ttgaagataa gaacgttaga 60
tttttacgtt tatgcttcac tgatattaac ggtactgaaa aggcagttga agtaccaact 120
agtcaattag ataaagtatt gaccaacgac attcgctttg acggatcatc aattgatgga 180
tttgttcgtc ttgaagaaag tgacatggtt ctatatccag acttttcaac ttggtcagta 240
ttaccatggg gtgatgaaca cggcggcaag atcggtcgtt tgatttgttc agttcacaca 300
actgatggta aagcttttgc aggtgatcca agaaataact tgaaacgagt tattggtgaa 360
atggaaaatg caggctttga tgcatttgac attggttttg aaatggaatt ccacctcttc 420
aagttagatg ataatggtaa ctggactact gaagttccag atcacgcttc atactttgat 480
atgacttcag atgatgaagg tgcacgctgc cgtcgtgaaa ttgttgaaac tttggaagat 540
atgggctttg aagttgaagc tgctcaccac gaagtaggtg atggtcaaca agaaattgac 600
tttagattcg acaatgcttt agcaactgct gatagatgcc aaacctttaa gatggttgct 660
cgcaccattg ctagaaaaca cggtttgttt gctacattta tggctaagcc tcttgaaggt 720
caagctggta acgggatgca caacaacatg tcactcttta agggtaagaa gaacgtattc 780
tacgacaaag atggtgaatt ccacctttca gatactgctc tttatttctt gaatggtatt 840
ttggaacatg ctcgtgcaat tactgcaatt ggtaacccaa ctgttaactc atacaagcgt 900
ttaattccag gttacgaagc accttgttac attgcttggg ctgctaagaa ccgttcacca 960
cttgttcgta ttccaagtgc tggtgaaatt aacactcgtt tggaaatgcg ttcagctgat 1020
ccaactgcta acccatactt attacttgct gcatgtttaa ctgctggttt aaacggtatt 1080
aaggaacaaa agatgccaat gaagccagtt gaagaaaaca tctttgaaat gactgaagaa 1140
gaaagagcaa agaagggtat taagccatta ccaactactc ttcacaacgc agttaaggca 1200
tttaaggaag atgatttaat taagagtgca ttaggtgatc acttaactcg cagctttatt 1260
gaatccaagg aattggaatg gtctaagtat tcacaatcag tttcagattg ggaacgtcaa 1320
cgttacatga actggtaa 1338
<210> 9
<211> 1338
<212> DNA
<213> Lactobacillus acidophilus
<400> 9
atgtccaaac agtacaccgc cgaggagatt aagaccgaag tcgaggataa gaatgtgcgc 60
tttctccgcc tgtgctttac cgacatcaac ggcaccgaga aagcagtgga ggtccctacc 120
tcccagctgg acaaggtcct gaccaacgac atccgcttcg atggctcttc tatcgacggc 180
ttcgtgcgcc tcgaagagtc cgacatggtc ctgtacccag atttctccac ctggtccgtg 240
ctgccatggg gcgatgaaca cggcggtaag atcggccgcc tcatctgttc cgtgcacacc 300
accgacggca aggcattcgc aggtgatcca cgcaacaacc tcaaacgcgt gatcggcgaa 360
atggaaaacg ccggcttcga tgccttcgat attggtttcg agatggagtt tcacctgttc 420
aagctggacg acaacggcaa ctggactacc gaggtgccag atcacgcatc ctacttcgac 480
atgacctctg atgatgaagg cgcacgctgc cgccgtgaga tcgtggagac cctggaggat 540
atgggtttcg aagtggaagc cgcccaccat gaagtcggcg atggccagca ggaaatcgac 600
ttccgcttcg ataacgccct ggcaaccgcc gatcgctgcc agaccttcaa gatggtggcc 660
cgcactattg cacgcaagca tggcctcttc gcaaccttca tggccaagcc actggagggt 720
caggccggta acggcatgca caacaatatg tctctcttca agggtaagaa aaacgtgttc 780
tacgacaagg atggcgagtt ccacctgtcc gacaccgccc tgtactttct gaacggcatc 840
ctggagcacg cccgcgcaat tactgccatc ggcaacccaa ccgtaaacag ctacaagcgc 900
ctcatccctg gttacgaagc accatgctat atcgcctggg ccgcaaagaa ccgctctcca 960
ctcgtgcgta tcccatccgc aggcgaaatc aacacccgcc tggaaatgcg ctccgcagat 1020
ccaaccgcca atccatacct gctgctcgcc gcatgcctca cggcggggct gaacggtatt 1080
aaggagcaga aaatgcctat gaagccagtg gaggagaaca tcttcgagat gaccgaagag 1140
gaacgcgcca agaagggcat caagccactg cctactaccc tccacaatgc cgtcaaagcc 1200
ttcaaggagg acgatctcat caagtccgca ctgggtgatc acctgacccg ctcttttatc 1260
gagtccaagg agctggagtg gtccaaatac tcccagtccg tgtccgactg ggaacgccaa 1320
cgctacatga attggtaa 1338
<210> 10
<211> 1082
<212> DNA
<213> E. coli W3110
<400> 10
atggctcagc tttcgttaca acatattcaa aaaatctacg ataaccaggt gcatgtggtg 60
aaggacttca acctggaaat tgccgataaa gagttcatcg tgtttgtcgg cccgtcgggc 120
tgcggtaagt cgaccaccct gcgcatgatt gccgggcttg aggagatcag cggcggcgat 180
ctgttgatcg acggcaaacg aatgaatgac gttccagcca aagcacgcaa tatagcgatg 240
gtgttccaga actacgcgtt gtatccgcat atgacggttt acgacaacat ggcgtttggt 300
ctgaagatgc aaaaaatcgc caaagaggtg attgatgagc gggtgaactg ggcggcgcaa 360
attctcggcc tgcgtgagta cctgaaacgt aagccggggg cgctttccgg cgggcaacgt 420
cagcgagtgg cgcttgggcg ggcgattgta cgcgaagcgg gcgtgttttt aatggatgaa 480
ccgctctcta accttgatgc caagctgcgc gtgcaaatgc gcgcagagat cagcaagctg 540
catcagaaac tgaacaccac catgatctac gtgacccacg atcagaccga agcgatgacc 600
atggcgacgc ggattgtgat tatgaaagac gggattgttc agcaagtagg tgcgccgaaa 660
accgtttata accaacccgc gaatatgttt gtttccggat ttattggatc accagcgatg 720
aattttattc gcggcacgat cgatggcgat aaattcgtta cggaaacgct taaattaacc 780
attcccgaag agaaattagc ggttctgaaa acacaggaaa gtttgcataa gcccatcgtg 840
atgggaatac gaccggaaga tattcatccg gacgcgcaag aggaaaataa catttccgcc 900
aaaattagcg tggcagaatt aaccggtgcg gaatttatgc tctacaccac ggttgggggc 960
acgagttagt ggtccgtgct ggtgcgttaa atgattatca tgcaggagaa aatatcacta 1020
ttcattttga tatgacgaaa tgtcatttct ttgatgcaga aacggaaata gcaattcgct 1080
aa 1082
Claims (10)
1. a kind of genetic engineering bacterium of high yield L-Trp, which is characterized in that the genetic engineering bacterium is the base in Escherichia coli
It is mutated because introducing trpE (S40F) in group, while the promoter of tryptophan operon is replaced with Ptrc promoters；It will be by
AroG (S180F) gene integrations of Ptrc promoters control are in the sites tyrR；Plac promoters are controlled serA (H344A,
N364A) gene integration is in yjiV pseudogenes site.
2. the genetic engineering bacterium of high yield L-Trp as described in claim 1, which is characterized in that the base described in claim 1
On the basis of engineering bacteria, the nucleotide sequence such as SEQ ID No that further control Plac promoters：Paddy ammonia shown in 9
Amide synthetase encoding gene glnA is integrated into ycjV pseudogenes site.
3. the genetic engineering bacterium of high yield L-Trp as claimed in claim 1 or 2, which is characterized in that the genetic engineering bacterium
Using E.coli W3110 as starting strain.
4. the genetic engineering bacterium of high yield L-Trp as claimed in claim 1 or 2, which is characterized in that the trpE (S40F)
The nucleotide sequence of gene such as SEQ ID No：Shown in 2；The nucleotide sequence such as SEQ ID No of aroG (S180F) gene：
Shown in 3；The nucleotide sequence such as SEQ ID No of serA (H344A, the N364A) gene：Shown in 5.
5. purposes of any genetic engineering bacteriums of claim 1-4 in fermenting and producing L-Trp.
6. purposes as claimed in claim 5, which is characterized in that carry out shake flask fermentation using the genetic engineering bacterium：
Seed liquor will be prepared after actication of culture, is inoculated into equipped in 500mL triangular flasks by 10-15% inoculum concentrations, nine layers of gauze seal
Mouthful, 37 DEG C, 200r/min shaken cultivations maintain pH in 7.0-7.2 in fermentation process by adding ammonium hydroxide；It is given instruction with phenol red
Agent, zymotic fluid color is considered as when no longer changing lacks sugar, and 1-2mL 60% (m/v) glucose solution is added when lacking sugar；Fermentation week
Phase 20-24h；
The fermentation medium group becomes：Glucose 20-40g/L, (NH4)2SO42-6g/L, KH2PO41-5g/L, MgSO4·
7H2O 0.5-2g/L, yeast extract 1-5g/L, FeSO4·7H2O 30-60mg/L, MnSO4·7H2O 1-5mg/L, VH
0.1-0.5mg/L, VB10.5-1.0mg/L, micro-mixed liquor 1-3ml/L, phenol red 15-30g/L, remaining is water, pH
7.0-7.2,115 DEG C of high steam pots sterilizing 15min.
7. purposes as claimed in claim 5, which is characterized in that carry out 5L ferment tanks using said gene engineering bacteria：
Seed liquor will be prepared after actication of culture, fresh fermentation medium is accessed according to 15-20% inoculum concentrations, start to ferment, sent out
It controls pH during ferment to stablize 7.0 or so, temperature maintains 37 DEG C, and dissolved oxygen is between 25-35%；When the grape in culture medium
After sugar consumption is complete, the glucose solution of stream plus 80% (m/v) maintain the concentration of glucose in fermentation medium in 0.1-5g/
L；Fermentation period 35-40h；
The fermentation medium group becomes：Glucose 20-40g/L, (NH4)2SO42-6g/L, KH2PO41-5g/L, MgSO4·
7H2O 0.5-2g/L, yeast extract 1-5g/L, FeSO4·7H2O 30-60mg/L, MnSO4·7H2O 1-5mg/L, VH
0.1-0.5mg/L, VB10.5-1.0mg/L, micro-mixed liquor 1-3ml/L, remaining is water, pH 7.0-7.2,115 DEG C of height
Press steam copper sterilizing 15min.
8. purposes as claimed in claims 6 or 7, which is characterized in that the micro-mixed liquor group is divided into：Na2MoO4·
2H2O 2.5g/L, AlCl3·6H2O 2.5g/L, NiSO4·6H2O 2.5g/L, CoCl2·6H2O 1.75g/L, CaCl2·
2H2O 10g/L, ZnSO4·7H2O 0.5g/L, CuCl2·2H2O 0.25g/L, H3BO3 0.125g/L。
9. a kind of construction method of the genetic engineering bacterium of high yield L-Trp, steps are as follows：
(1) tryptophan is grasped using E.coli W3110 as starting strain using Escherichia coli CRISPR/Cas gene editing technologies
It indulges while sub promoter replaces with Ptrc promoters and introduces trpE (S40F) gene, the nucleosides of trpE (S40F) gene
Acid sequence such as SEQ ID No：Shown in 2；
(2) aroG (S180F) gene integrations controlled Ptrc promoters to the sites tyrR, aroG (S180F) gene
Nucleotide sequence such as SEQ ID No：Shown in 3；
(3) serA (H344A, the N364A) gene integrations controlled Plac promoters are to yjiV pseudogenes site, the serA
The nucleotide sequence of (H344A, N364A) gene such as SEQ ID No：Shown in 5.
10. a kind of construction method of the genetic engineering bacterium of high yield L-Trp as claimed in claim 9, which is characterized in that
On the basis of the genetic engineering bacterium that claim 9 is built, the glutamine synthelase for further controlling Plac promoters encodes
Gene glnA is integrated into ycjV pseudogenes site, the nucleotide sequence such as SEQ ID No of the glnA genes：Shown in 9.
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| CN110438058A (en) * | 2019-08-28 | 2019-11-12 | 宁夏伊品生物科技股份有限公司 | A kind of recombinant bacterial strain producing L-Trp and its construction method and application |
| RU2806731C1 (en) * | 2019-09-03 | 2023-11-03 | Нинся Эппэнь Биотек Ко., Лтд | Application of transporter carrier gene that improves l-tryptophan production efficiency in escherichia coli |
| WO2021042460A1 (en) | 2019-09-03 | 2021-03-11 | 宁夏伊品生物科技股份有限公司 | Application of transport carrier gene which improves l-tryptophan production efficiency in escherichia coli |
| JP2022547432A (en) * | 2019-09-03 | 2022-11-14 | 寧夏伊品生物科技股▲ふん▼有限公司 | Use of transporter gene in Escherichia coli to improve L-tryptophan production efficiency |
| JP7475434B2 (en) | 2019-09-03 | 2024-04-26 | 寧夏伊品生物科技股▲ふん▼有限公司 | Use of transporter genes in Escherichia coli to improve L-tryptophan production efficiency |
| CN114729302A (en) * | 2019-10-31 | 2022-07-08 | 大象株式会社 | Strain having improved amino acid-producing ability due to inactivation of GLSB gene and method for producing the same |
| CN114729302B (en) * | 2019-10-31 | 2024-03-26 | 大象株式会社 | Strain having improved amino acid-producing ability due to inactivation of GLSB gene and method for producing the same |
| CN111004761A (en) * | 2019-12-02 | 2020-04-14 | 天津科技大学 | L-tyrosine gene engineering bacterium, method for producing L-tyrosine by using same and application thereof |
| KR102312197B1 (en) * | 2020-06-18 | 2021-10-13 | 경희대학교 산학협력단 | Production of tryptophan from methane by metabolic engineered methanotrophs |
| CN111926002A (en) * | 2020-09-16 | 2020-11-13 | 中国科学院天津工业生物技术研究所 | TrpE mutant and application thereof in gene engineering bacteria for producing L-tryptophan |
| CN114085801A (en) * | 2021-11-29 | 2022-02-25 | 江南大学 | Recombinant escherichia coli for producing L-tryptophan and application thereof |
| CN114085801B (en) * | 2021-11-29 | 2023-08-08 | 江南大学 | Recombinant escherichia coli for producing L-tryptophan and application thereof |
| CN115948311A (en) * | 2022-09-26 | 2023-04-11 | 天津科技大学 | Genetically engineered bacterium for producing 5-hydroxytryptophan and construction method and application thereof |
| CN115895976B (en) * | 2022-12-20 | 2024-02-06 | 哈尔滨象柏生物科技有限公司 | Escherichia coli for producing L-tryptophan and application thereof in producing L-tryptophan |
| CN115895976A (en) * | 2022-12-20 | 2023-04-04 | 黑龙江金象生化有限责任公司 | Escherichia coli for producing L-tryptophan and application of escherichia coli for producing L-tryptophan |
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