CN108753860B - The building of Recombinant organism and its purposes of production L-Trp - Google Patents
The building 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 utilize the method for 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 up to 40-45g/L in 5L ferment tank 35-40h, 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 technique
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 is used, this method passes through fermenting and producing L- color ammonia by substrate of low price raw materials such as glucose
Acid, lower production costs, production technology are relatively easy controllable.
Since the metabolic pathway of synthesizing of L-Trp is long and complex, related 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 constructs 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 constructing 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, which is typically chosen, to be passed through
Building 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 so can achieve the purpose for strengthening L-Trp route of synthesis, but there is also carry resistance, need to add for constructed engineered strain
Inducer brings the adverse effect such as burden to thalli growth.Chen L and Zeng AP (Applied Microbiology&
Biotechnology.2017.101 (2): the Escherichia coli L- for not carrying plasmid 559) is constructed by the method for gene integration
Tryptophan engineering bacteria analyzes outer intermediate product and the precursor intracellular variation in its fermentation process, mentions after fermentation in text
Phase, L-Glutamine content intracellular was lower, 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 decline even inactivates, 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 lower 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, matter in fermentation process mostly
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 period 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 lower, extraction process is complicated.
Summary of the invention
For above-mentioned there are problem, object of the present invention is to the method buildings 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 and promoter are replaced) by metabolic engineering, to the L- of Escherichia coli
Related gene is combined obtained from transformation in tryptophan synthetic pathway.Base relevant to L-Trp synthesis in Escherichia coli
Because including aroG (3- deoxidation-σ-Arab's heptanone saccharic acid -7- phosphate synthase encoding gene), serA, (3-phoshoglyceric acid is de-
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 sequence
Using the generally acknowledged IUPAC nomenclature of amino acid residue, with a letter abbreviations form.DNA nucleic acid sequence is using generally acknowledged
IUPAC nomenclature.
2, the mark of mutant
The amino acid being mutated in mutant is indicated using " amino acid of Original amino acid position replacement ".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 solution of the present invention is summarized as follows:
One of technical solution of the present invention: being the genetic engineering bacterium for constructing 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
To introduce trpE (S40F) mutation while Ptrc promoter；AroG (S180F) gene integration that will be controlled by Ptrc promoter
To the site tyrR；SerA (H344A, the N364A) gene integration that Plac promoter is controlled is to yjiV pseudogene 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 technology is 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 promoter, strengthen color ammonia
The feedback inhibition of key enzyme anthranilate synthase is released while sour operon synthetase series；
(2) aroG (S180F) gene integration for the solution feedback inhibition for controlling Ptrc promoter is strengthened and is closed to the site tyrR
Its feedback inhibition is released while key enzyme 3- deoxy-arabinose heptanone saccharic acid -7- phosphate synthase；
(3) serA (H344A, the N364A) gene integration controlled Plac promoter is to yjiV pseudogene site, before increase
Body object Serine releases its feedback inhibition while synthesis.
The nucleotide sequence of the Ptrc promoter is as shown in SEQ ID No:1.
The nucleotide sequence of trpE (S40F) gene is as shown in SEQ ID No:2.
The nucleotide sequence of aroG (S180F) gene is as shown in SEQ ID No:3.
The nucleotide sequence of the Plac promoter is as shown in SEQ ID No:4.
The nucleotide sequence of serA (H344A, the N364A) gene is as shown in SEQ ID No:5.
The nucleotide sequence of the tyrR gene is as shown in SEQ ID No:6.
The nucleotide sequence of the yjiV gene is as shown in SEQ ID No:7.
The two of technical solution of the present invention: being the genetic engineering bacterium of one plant of fast and efficient production L-Trp of further building
E.coli TRP04, the genetic engineering bacterium are introduced on the basis of genetically engineered E.coli TRP03 from acidophilus cream
The glutamine synthelase encoding gene glnA of bacillus (Lactobacillus acidophilus)；It further, is by Plac
(nucleotide sequence of optimization front and back is respectively such as SEQ ID No:8 and SEQ ID for glnA gene after the optimization of promoter control
Shown in No:9) it is integrated into ycjV pseudogene site (nucleotide sequence of the ycjV gene is as shown in SEQ ID No:10), increase
Add the supply of precursor L-Glutamine.
The three of technical solution of the present invention: being 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 triangle equipped with fermentation medium is inoculated by 10-15% inoculum concentration
In bottle, the sealing of nine layers of gauze, 37 DEG C, 200r/min shaken cultivation maintains pH in 7.0- in fermentation process by adding ammonium hydroxide
7.2；Adding 60% (m/v) glucose solution maintains fermentation (give instruction agent with phenol red, when fermentation liquid color no longer changes
It is considered as and lacks sugar, add 1-2mL60% (m/v) glucose solution when lacking sugar)；Fermentation period 20-24h.
Preferred fermentation medium composition are as follows: 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 component are as follows: 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 fermentation 20-24h,
The yield of L-Trp is up to 10-12g/L after E.coli TRP04 shake flask fermentation 20-24h.
It is as follows that 5L ferment tank is 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 concentration, 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 composition are as follows: 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 component are as follows: 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 up to 40-45g/L using 5L ferment tank 35-40h.
The invention has the advantages that and the utility model has the advantages that
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 convenience 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 optimizing from the glutamine synthetase gene of lactobacillus acidophilus
Sour engineered strain improves the yield of L-Trp significantly, and the gene is relevant applied to L-Glutamine synthesis is related to
The building 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 tank 40h, compared to the industrialization L- color for carrying plasmid
Propylhomoserin production bacterial strain improves 15.1%, shows the potentiality of industrialized production of L-tryptophan.
Detailed description of the invention
Fig. 1: (a) pREDCas9 plasmid map, (b) pGRB plasmid map.
The building of Fig. 2: Ptrc trpE (S40F) gene replacement segment and verifying 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: former
Bacterium control.
The building of Fig. 3: Ptrc aroG (S180F) gene integration segment and verifying 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 control；
6: positive bacteria identifies segment.
The building of Fig. 4: Plac serA (H344A, N364A) gene integration segment and verifying 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.
The building of Fig. 5: Plac glnA gene integration segment and verifying electrophoretogram.Wherein: M:1kb DNA marker；1: on
Swim homology arm；2:glnA genetic fragment；3 downstream homology arms；4: overlapping fragments；5: opportunistic pathogen control；6: positive bacteria identifies segment.
Fig. 6: HPLC measurement L-Trp standard curve.
Fig. 7: different strains shake flask fermentation L-Trp yield.
Fig. 8: 5L fermentor L-Trp resultant curve.
Specific embodiment
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, to the various changes or change of material component and dosage progress in these embodiments
It belongs to the scope of protection of the present invention.
The percentage sign " % " being related in embodiment refers to percent by volume if not specified；The percentage of solution
" % (m/v) " refers to the grams in 100mL solution containing solute.
Embodiment 1: building 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, 2015,31:13-21.) it carries out, two plasmid maps used in this method are shown in
Attached drawing 1.Wherein pREDCas9 carries the elimination system of gRNA expression plasmid pGRB, the Red recombination system and Cas9 egg of λ bacteriophage
White expression system, miramycin resistance (working concentration: 100mg/L), 32 DEG C of cultures；PGRB is using pUC18 as skeleton, including promoter
The bond area J23100, gRNA-Cas9 sequence and terminator sequence, amicillin resistance (working concentration: 100mg/L), 37
DEG C culture.
Specific step is as follows for this method:
1.1 pGRB plasmid constructions
Building plasmid pGRB purpose be in order to transcribe corresponding gRNA, thus with Cas9 albumen formed complex, and
By base pairing and PAM identifying purpose gene target site, target DNA double-strand break is realized.Using the DNA piece comprising target sequence
Section and the method for the carrier segments recombination of linearisation construct pGRB plasmid.
1.1.1 target sequence designs
Target sequence (PAM:5 '-NGG-3 ') is designed using CRISPR RGEN Tools
1.1.2 the preparation of the DNA fragmentation comprising target sequence
Design primer: 5 '-linearized vector end sequences (15bp)-restriction enzyme site-target sequence (not including PAM sequence)-
The primer of linearized vector end sequence (15bp) -3 ' and its reverse complemental includes target sequence by the annealing preparation of single stranded DNA
DNA fragmentation.Reaction condition: 95 DEG C of initial denaturation, 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, mix gently rear ice bath 20min, 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 is centrifuged 2min, abandons part
Supernatant is applied to the plate containing 100mg/L ampicillin after staying 200 μ L or so that thallus is resuspended, plate is inverted, in 37
It DEG C is incubated overnight.It is identified after plate grows single colonie by bacterium colony PCR, selects positive recombinant.
1.1.6 clone identification
PCR positive bacterium colony is seeded in the LB culture medium containing 100mg/L ampicillin after being incubated overnight and protects bacterium, mentioned
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 to the upper of integration site
Downstream sequence is template, is designed upstream and downstream homology arm primer (amplification length about 400-500bp)；Using to 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 preparation and reorganization segment.Archaeal dna polymerase used in PCR is purchased from TaKaRa company, the PrimeSTAR HS including high-fidelity
DNA Polymerase and PCR product carry the system and method for Ex.taq the DNA Polymerase, PCR of cohesive end
It is as follows:
HS enzyme PCR amplification system
Ex.taq enzyme PCR amplification system
The system of over-lap PCR is as follows:
HS enzyme over-lap PCR amplification system
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 system
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 condition: initial denaturation (95 DEG C) 5min；Then carry out 30 wheel circulations: denaturation (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 goes to W3110 is turned in competence using the method that electricity turns, thallus is recovered and is cultivated
It is coated on the LB plate containing miramycin afterwards, 32 DEG C are incubated overnight.Single colonie is grown in resistant panel carries out bacterium with identification primer
PCR is fallen, positive recombinant is screened.
1.3.2 the purpose bacterial strain electrotransformation competence preparation 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 plasmid
Expression.Culture medium needed for prepared by competence and preparation process are operated referring 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 thallus of culture of recovering after change is coated on the LB plate containing ampicillin and miramycin, and 32 DEG C are incubated overnight.It is same with upstream
The downstream primer of source arm upstream primer and downstream homology arm, or the special identification primer of design, carry out bacterium colony PCR verifying, 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 medium containing 0.2% arabinose and is incubated overnight, is coated with after appropriate dilution
In on the LB plate 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 colonie of miramycin resistant panel growth protects bacterium.
1.4.2 the elimination of pREDCas9 plasmid
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 plate of non-resistant, 37 DEG C are incubated overnight.To LB plate of the point containing miramycin resistance and non-resistant, miramycin is selected
Resistant panel is not grown, and the single colonie of non-resistant plated growth protects bacterium.
The genetic engineering of 2.L- tryptophan engineered strain E.coli TRP03 combines transformation process
TrpE (S40F) mutation is introduced while 2.1 tryptophan operon promoters are replaced
2.1.1 carrier T of the building containing trpE (S40F) mutation
Using E.coli W3110 (ATCC27325) genome as template, according to trpE gene 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
Sequence (TCC sports TTT) and reverse complemental, and with its upstream and downstream homology arm segment of Ex.taq enzyme PCR amplification.Above-mentioned segment is logical
The method for crossing Ex.taq enzyme over-lap PCR obtains the segment (upstream homology arm-downstream homology arm) containing trpE (S40F) mutation.By institute
Segment and carrier T 4:1 mixing in molar ratio (total amount is no more than 100ng) are obtained, the solution I ligase of 5 μ L is added, adds water
It mends to 10 μ L systems, after 16 DEG C of connection 1h, selects positive transformant by the operation of method shown in 1.1.5, extract plasmid and send sequencing really
It is fixed successfully to construct T load-trpE (S40F).
2.1.2 tryptophan operon is transformed
Using E.coli W3110 (ATCC27325) genome as template, it is same that upstream is designed according to trpE gene upstream sequence
Source arm primer (trpE (S40F) -1, trpE (S40F) -5)；It is same according to sequence design downstream with T load-trpE (S40F) for template
Source arm primer (trpE (S40F) -6, trpE (S40F) -4), wherein the design addition of trc promoter sequence is in primer trpE
(S40F) on -5 and trpE (S40F) -6, with its upstream and downstream homology arm segment of HS enzyme PCR amplification.Above-mentioned segment is overlapped by HS enzyme
(upstream homology arm-Ptrc trpE (S40F)-downstream is same for the replacement segment of method acquisition Ptrc trpE (S40F) gene of PCR
Source arm).Constructing 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.Preparation
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 of the PCR verifying of the building and positive strain of Ptrc trpE (S40F) gene replacement 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, and when PCR is verified, identification primer (trpE (S40F) -7) design is existed
Inside Ptrc promoter sequence, 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 site genome tyrR
2.2.1 carrier T of the building containing aroG (S180F) mutation
Using E.coli W3110 (ATCC27325) genome as template, according to aroG gene 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 is same
When contain mutant nucleotide sequence (TCT sports TTT) and reverse complemental, and with its upstream and downstream homology arm segment of Ex.taq enzyme PCR amplification.On
Segment is stated by the method for Ex.taq enzyme over-lap PCR to obtain the segment containing aroG (S180F) mutation (upstream homology arm-downstream is same
Source arm).By obtained segment and carrier T, 4:1 mixes (total amount is no more than 100ng) in molar ratio, and the solution I that 5 μ L are added connects
Enzyme is connect, water is added to mend to 10 μ L systems, after 16 DEG C of connection 1h, positive transformant is selected by the operation of method shown in 1.1.5, extracts plasmid
Sequencing is sent to determine successfully building T load-aroG (S180F).
2.2.2 the integration of Ptrc aroG (S180F) gene
Using E.coli W3110 (ATCC27325) genome as template, according in the upstream and downstream sequence design of tyrR gene
Homology arm primer (tyrR-1, tyrR-2) and downstream homology arm primer (tyrR-3, tyrR-4) are swum, with the T load-aroG of building
It (S180F) is template, according to the primers (aroG (S180F) -5, aroG (S180F) -6, wherein trc of aroG gene
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 PCR
Homology arm-Ptrc aroG (S180F)-downstream homology arm).The DNA fragmentation containing target sequence that building 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
The operation of method shown in 1.4, it is final to obtain bacterial strain E.coli TRP02.The building of Ptrc aroG (S180F) gene integration segment
Attached drawing 3 is seen with the PCR of the positive strain electrophoretogram 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 site genome yjiV
2.3.1 carrier T of the building containing serA (H344A, N364A) mutation
Using E.coli W3110 (ATCC27325) genome as template, according to serA gene 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
It swims homology arm primer (serA (H344A) -3, serA (H344A) -4), wherein serA (H344A) -2, serA (H344A) -3 is simultaneously
Containing mutant nucleotide sequence (CAC sports GCC) and reverse complemental, and with its upstream and downstream homology arm segment of Ex.taq enzyme PCR amplification.It is above-mentioned
The method that segment passes through Ex.taq enzyme over-lap PCR obtains the segment containing serA (H344A) mutation, and (upstream homology arm-downstream is homologous
Arm).By obtained segment and carrier T, 4:1 mixes (total amount is no more than 100ng) in molar ratio, and the solution I connection of 5 μ L is added
Enzyme adds water to mend to 10 μ L systems, after 16 DEG C of connection 1h, selects positive transformant by the operation of method shown in 1.1.5, extracts plasmid and send
Sequencing determines successfully building T load-serA (H344A).
With T load-serA (H344A) for template, it is mutated according to serA gene 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 contains mutant nucleotide sequence simultaneously
(AAC sports GCC) and reverse complemental, and with its upstream and downstream homology arm segment of Ex.taq enzyme PCR amplification.Above-mentioned segment passes through
The method of Ex.taq enzyme over-lap PCR obtains the segment containing serA (H344A, N364A) mutation, and (upstream homology arm-downstream is homologous
Arm).By obtained segment and carrier T, 4:1 mixes (total amount is no more than 100ng) in molar ratio, and the solution I connection of 5 μ L is added
Enzyme adds water to mend to 10 μ L systems, after 16 DEG C of connection 1h, selects positive transformant by the operation of method shown in 1.1.5, extracts plasmid and send
Sequencing determines successfully building T load-serA (H344A, N364A).
2.3.2 the integration of Plac serA (H344A, N364A) gene
Using E.coli W3110 (ATCC27325) genome as template, according in the upstream and downstream sequence design of yjiV gene
Swim homology arm primer (yjiV-1, yjiV-2) and downstream homology arm primer (yjiV-3, yjiV-4)；With T load-serA (H344A,
It N364A is) template, according to its primers (serA (H344A, N364A) -1, serA (H344A, N364A) -2),
In, the design addition of lac promoter sequence is on primer yjiV-2 and serA (H344A, N364A) -1, with HS enzyme PCR amplification Plac
SerA (H344A, N364A) genetic fragment.Above-mentioned segment by the method for HS enzyme over-lap PCR obtain Plac serA (H344A,
N364A) the integration segment (upstream homology arm-Plac serA (H344A, N364A)-downstream homology arm) of gene.Construct 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.Preparation
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 verifying of the building 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. primer involved in during strain improvement see the table below:
Embodiment 2: the glutamine synthelase for coming from lactobacillus acidophilus (Lactobacillus acidophilus) is introduced
(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 codon software common in Escherichia coli, (codon is excellent
Sequence before and after changing is respectively as shown in SEQ ID NO:8 and SEQ ID NO:9), allow to the efficient transcription in Escherichia coli.
Sequence after optimization is sent to Jin Weizhi company to synthesize, obtains the recombinant plasmid pUC57glnA with glnA gene, wherein enzyme
Enzyme site is Hind III and BamH I, is stored in Escherichia coli.
2.Plac glnA is integrated into genome ycjV pseudogene site
Using E.coli W3110 (ATCC27325) genome as template, according to the upstream and downstream sequence design of ycjV pseudogene
Upstream homology arm primer (ycjV-1, ycjV-2), downstream homology arm primer (ycjV-3, ycjV-4), with recombinant plasmid
PUC57glnA is template, is expanded according to its sequence design Plac glnA fragment primer (glnA-1, glnA-2), and with HS enzyme PCR
Increase its upstream and downstream homology arm segment and Plac glnA segment.Above-mentioned segment obtains Plac by the method for HS enzyme over-lap PCR
The integration segment (the upstream downstream homology arm-Plac glnA- homology arm) of glnA gene.The sequence containing target that building pGRB-ycjV is used
The DNA fragmentation of column 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 integration
The electrophoretogram of the PCR verifying of the building 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 fragment should be 1414bp, Plac glnA gene integration
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
Increasing fragment length should be 1859bp.
Embodiment 3: L-Trp is produced using Recombinant organism shake flask fermentation
A kind of concrete operations for carrying out shake flask fermentation production L-Trp using Recombinant organism are as follows:
Inclined-plane culture: taking -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 is scraped with oese and is inoculated in the 500mL equipped with 30mL seed culture medium
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 flask equipped with fermentation medium by 10-15% (v/v) inoculum concentration
(final volume 30mL), the sealing of nine layers of gauze, 37 DEG C, 200r/min shaken cultivation is maintained by adding ammonium hydroxide in fermentation process
PH is in 7.0-7.2；Adding 60% (m/v) glucose solution maintains fermentation (give instruction agent with phenol red, fermentation liquid 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 fermentation liquid
For initial value 20-40g/L)；Fermentation period 22-26h；
L-Trp concentration mensuration in fermentation liquid: collecting fermentation liquid 1mL, and 13000rpm is centrifuged 1min, collects supernatant.Make
It is diluted to certain multiple (being diluted to 0.1-0.5g/L) by supernatant is collected with deionized water, uses liquid after 0.22 μm of micro porous filtration
Mutually detection L-Trp content, chromatographic condition are as follows: chromatographic column Kromasil C18 column (250mm × 460mm, 5 μm), mobile phase
For 10% acetonitrile solution, flow velocity 1.0mL/min, 40 DEG C of column temperature, Detection wavelength 278nm, sample volume is 20 μ L, and appearance time is about
The concentration of L-Trp in fermentation liquid is calculated according to its peak area by the standard curve of drafting by 3.752min；
The drafting of L-Trp standard curve: taking L-Trp concentration respectively is 0.1g/L, 0.2g/L, 0.3g/L, 0.4g/
The solution of L, 0.5g/L, according to the corresponding peak area of L-Trp that above-mentioned chromatographic condition obtains respective concentration 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 as follows: 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 component are as follows: 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 component are as follows: 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 component are as follows: 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 building,
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 save
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, experimental result are as shown in Figure 7.Starting strain E.coli W3110 hardly accumulates L-Trp, engineered to combine
The L-Trp engineered strain E.coli TRP03 accumulation L-Trp arrived has not only reached accumulation L- up to 8.75 ± 0.75g/L
The purpose of tryptophan, have also exceeded carry plasmid L-Trp engineered strain E.coli TRTH accumulation L-Trp 6.75 ±
The yield of 0.84g/L, and introduce the product of the L-Trp engineered strain E.coli TRP04 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 tank
1. as follows using a kind of concrete operations that Escherichia coli progress 5L fermentor normally produces L-Trp:
Slant activation culture: protecting in tube from -80 DEG C of refrigerators and scrape a ring strain, is 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: taking appropriate amounts of sterilized water in eggplant-shape bottle, and bacteria suspension is accessed in seed culture medium, and 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: accessing fresh fermentation medium according to 15-20% inoculum concentration, starts to ferment, control 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 as follows: 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 component are as follows: 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 component are as follows: 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 component are as follows: 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 fermentor shaking flask is 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 control, and experimental result is as schemed
Shown in 8.The concentration of L-Trp in fermentor is monitored by the method for L-Trp concentration in above-mentioned shake flask fermentation detection fermentation liquid,
E.coli TRP04 fermentation 40h can accumulate L-Trp up to 44.1g/L, improve 15.1% compared to E.coli TRTH.
Although above-mentioned have been disclosed the preferable embodiment of the present invention, it is not intended to limit the invention, any ability
Field technique personnel can be various change and modification, therefore guarantor of the 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>purposes of the building of Recombinant organism and its 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 (6)
1. a kind of genetic engineering bacterium of high yield L-Trp, which is characterized in that the genetic engineering bacterium is in Escherichia coli W3110
Genome on, trpE (S40F) mutation is introduced while the promoter of tryptophan operon is replaced with Ptrc promoter；It will
AroG (S180F) gene integration controlled by Ptrc promoter is in the site tyrR；Plac promoter is controlled serA (H344A,
N364A) gene integration is in yjiV pseudogene site；The nucleotide sequence that further Plac promoter is controlled such as SEQ ID
Glutamine synthelase encoding gene glnA shown in No:9 is integrated into ycjV pseudogene site；TrpE (S40F) gene
Nucleotide sequence is as shown in SEQ ID No:2；The nucleotide sequence of aroG (S180F) gene is as shown in SEQ ID No:3；
The nucleotide sequence of serA (H344A, the N364A) gene is as shown in SEQ ID No:5.
2. purposes of the genetic engineering bacterium described in claim 1 in fermenting and producing L-Trp.
3. purposes as claimed in claim 2, 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 by 10-15% inoculum concentration equipped in 500 mL triangular flasks, nine layers of gauze seal
Mouthful, 37 DEG C, 200 r/min shaken cultivations maintain pH in 7.0-7.2 in fermentation process by adding ammonium hydroxide；It is done and is referred to phenol red
Show agent, fermentation liquid color is considered as when no longer changing lacks sugar, adds 1-2 mL 60%(m/v when lacking sugar) glucose solution；Fermentation
Period 20-24 h；
The fermentation medium composition are as follows: glucose 20-40 g/L, (NH4)2SO42-6 g/L, KH2PO41-5 g/L,
MgSO4·7H2O 0.5-2 g/L, yeast extract 1-5 g/L, FeSO4·7H2O 30-60 mg/L, MnSO4·7H2O 1-5
Mg/L, VH 0.1-0.5 mg/L, VB1 0.5-1.0 mg/L, micro-mixed liquor 1-3 ml/L, phenol red 15-30 g/L,
Yu Weishui, pH 7.0-7.2,115 DEG C of 15 min of high steam pots sterilizing.
4. purposes as claimed in claim 2, which is characterized in that carry out 5 L 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 concentration, start to ferment, fermented
Control pH stablizes 7.0 or so in the process, and temperature maintains 37 DEG C, and dissolved oxygen is between 25-35%；When the glucose in culture medium
After ruing out of, the glucose solution of stream plus 80% (m/v) maintains the concentration of glucose in fermentation medium in 0.1-5 g/L；
Fermentation period 35-40 h；
The fermentation medium composition are as follows: glucose 20-40 g/L, (NH4)2SO42-6 g/L, KH2PO41-5 g/L,
MgSO4·7H2O 0.5-2 g/L, yeast extract 1-5 g/L, FeSO4·7H2O 30-60 mg/L, MnSO4·7H2O 1-5
Mg/L, VH 0.1-0.5 mg/L, VB1 0.5-1.0 mg/L, micro-mixed liquor 1-3 ml/L, remaining is water, pH 7.0-
7.2,115 DEG C of 15 min of high steam pots sterilizing.
5. purposes as described in claim 3 or 4, which is characterized in that the micro-mixed liquor component are as follows: Na2MoO4·
2H2O 2.5 g/L, AlCl3·6H2O 2.5 g/L, NiSO4·6H2O 2.5g/L, CoCl2·6H2O 1.75 g/L, CaCl2·
2 H2O 10g/L, ZnSO4·7 H2O 0.5 g/L, CuCl2·2 H2O 0.25 g/L, H3BO3 0.125g/L。
6. a kind of construction method of the genetic engineering bacterium of high yield L-Trp, steps are as follows:
(1) Escherichia coli CRISPR/Cas gene editing technology is used, using E. coli W3110 as starting strain, by tryptophan
The promoter of operon introduces trpE (S40F) gene, the core of trpE (S40F) gene while replacing with Ptrc promoter
Nucleotide sequence is as shown in SEQ ID No:2；
(2) aroG (S180F) gene integration controlled Ptrc promoter to the site tyrR, aroG (S180F) gene
Nucleotide sequence is as shown in SEQ ID No:3；
(3) serA (H344A, the N364A) gene integration controlled Plac promoter is to yjiV pseudogene site, the serA
The nucleotide sequence of (H344A, N364A) gene is as shown in SEQ ID No:5；
(4) the glutamine synthelase encoding gene glnA that Plac promoter controls is integrated into ycjV pseudogene site, it is described
The nucleotide sequence of glnA gene is as shown in SEQ ID No:9.
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| 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 |
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| CN110438058B (en) * | 2019-08-28 | 2021-10-26 | 宁夏伊品生物科技股份有限公司 | Recombinant strain for producing L-tryptophan and construction method and application thereof |
| US20220411835A1 (en) | 2019-09-03 | 2022-12-29 | Ningxia Eppen Biotech Co., Ltd | Application of transport carrier gene which improves l-tryptophan production efficiency in escherichia coli |
| KR102283626B1 (en) * | 2019-10-31 | 2021-08-02 | 대상 주식회사 | Strain with Improved Aromatic Amino Acid Production Capacity by glsB Gene Inactivation |
| 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 |
| CN111926002B (en) * | 2020-09-16 | 2021-01-05 | 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 |
| 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 |
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