CN109266592A - A kind of l-tyrosine genetic engineering bacterium and its method for producing l-tyrosine - Google Patents
A kind of l-tyrosine genetic engineering bacterium and its method for producing l-tyrosine Download PDFInfo
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Abstract
A kind of method that the present invention discloses l-tyrosine genetic engineering bacterium and its produces l-tyrosine, the genetic engineering bacterium has knocked out L-phenylalanine synthase gene pheA and its controlling gene pheL, the T7 phage rna polymerase that xylose promoter PxylF is controlled is integrated into the site lacZ, tyrA (the M53I that will be controlled by T7 promoter, A354V)-tyrB is integrated into the site tyrR, and the aroG (S180F) of T7 promoter control is integrated into yghX pseudogene site.L-tyrosine 6.8g/L can be accumulated for 24 hours by carrying out shake flask fermentation using the bacterial strain, in 5L fermentation cylinder for fermentation 30h yield up to 37.8g/L.The genetic engineering bacterium does not carry plasmid, addition antibiotic is not needed in fermentation process, simultaneously using the high efficient expression of xylose induction key gene, reduce production cost, avoid injury of the inducers such as IPTG to cell, the method of the production l-tyrosine is simple, controllability is strong, is suitable for industrialization large-scale production.
Description
Technical field
The present invention relates to technical field of bioengineering more particularly to a kind of l-tyrosine genetic engineering bacterium and its production L- junket
The method of propylhomoserin.
Background technique
L-tyrosine (L- β-p-hydroxyphenyl-Beta-alanine；(2S, 3R) -2- amino -3- p-hydroxyphenyl propionic acid) belong to fragrance
Race's amino acid is the conditionally essential amino acid of human body, it frequently as nutritional supplement and L-3,4 dihydroxyphenylalanine, p-Coumaric Acid, to hydroxyl
The raw material for preparing of the medication chemistries product such as base styrene is widely used in the industries such as food, feed, medicine and chemical industry.L-tyrosine
Production method include albumen hydrolysis, chemical synthesis, enzyme process and direct fermentation.Traditional l-tyrosine production method master
If albumen hydrolysis and enzyme process, but these methods are there are shortcomings, as raw material sources are limited, in reaction process enzymatic activity and
Stability is poor, technique is cumbersome, product composition is complicated etc..And microorganism direct fermentation production amino acid has raw material sources wide
General, the reaction advantages such as mild, easy to operate.Conjunction with the continuous development of metabolic engineering technology, to l-tyrosine in microorganism
Being rationally designed and being optimized at approach becomes research hotspot with the fermenting and producing for realizing l-tyrosine.
Currently, the route of synthesis and regulatory mechanism of l-tyrosine in Escherichia coli and corynebacterium glutamicum to study most
To be clear, metabolic pathway, which is transformed, to be concentrated mainly on the regulation of releasing approach, increases precursor supply, competition approach and adjusting is blocked to turn
Several aspects such as fortune system.Although the reported research about l-tyrosine metabolic engineering has sufficiently illustrated its metabolism way
Key gene, Regulation Mechanism and transformation target spot in diameter etc., but the fermenting and producing of l-tyrosine still remains low output or saccharic acid
The low problem of conversion ratio, and carry out there is presently no mature fermentation method production technology the industrialized production of l-tyrosine.
Summary of the invention
Technical problem to be solved by the present invention lies in provide a kind of l-tyrosine genetic engineering bacterium.
Another technical problem to be solved by this invention is to provide a kind of utilization said gene engineering bacteria production L- junket ammonia
The method of acid.
In order to solve the above technical problems, technical solution of the present invention first is that:
A kind of l-tyrosine genetic engineering bacterium, the genetic engineering bacterium are the methods by metabolic engineering in Escherichia coli
L-tyrosine synthesis related gene carries out corresponding transformation, blocks competition approach phenylalanine route of synthesis, releases the anti-of key enzyme
Feedback inhibits and transcription negative regulation, and the RNA polymerase of T7 bacteriophage is derived from using xylose promoter induction, starts by force in conjunction with T7
The high efficient expression of subsystem starting key gene.
The genetic engineering bacterium has knocked out L-phenylalanine synthase gene pheA and its controlling gene pheL, and xylose is opened
Mover PxylF control T7 phage rna polymerase be integrated into the site lacZ, by the tyrA controlled by T7 promoter (M53I,
A354V)-tyrB is integrated into the site tyrR, and the aroG (S180F) of T7 promoter control is integrated into yghX pseudogene site.
Technical solution of the present invention second is that: the construction method of said gene engineering bacteria, the specific steps are as follows:
(1) Escherichia coli CRISPR/Cas9 gene editing technology is used, using E.coli W3110 as starting strain, is knocked out
PheLA gene；
(2) junction fragment of xylose promoter PxylF and t7 rna polymerase T7RNAP are constructed, and is integrated into lacZ
Site；
(3) junction fragment of PT7 promoter and tyrA (M53I, A354V)-tyrB is constructed, and is integrated into tyrR
Point.
(4) junction fragment of PT7 promoter and aroG (S180F) is constructed, and is integrated into yghX pseudogene site；
Technical solution of the present invention third is that: utilize said gene engineering bacteria production l-tyrosine method.
Fermented and cultured is carried out by the inoculum concentration of 10-15%, addition 10g/L xylose induces target gene table when fermentation starts
It reaches, indicator is made with phenol red in fermentation process, control pH in 7.0-7.2 by adding ammonium hydroxide, chignon adds grape in the process
Sugar juice.
Preferred fermentation medium composition are as follows: glucose 10g/L, xylose 10g/L, yeast powder 5g/L, (NH4)2SO4 5g/
L, KH2PO43g/L, MgSO4·7H2O 2g/L, sodium citrate 2g/L, CaCl2·2H2O 0.015g/L, FeSO4·7H2O
30mg/L, micro-mixed liquor (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,
H3BO30.125g/L) 1.5mL/L, VB10.5mg/L, VH0.5mg/L, L-phenylalanine 0.5g/L, pH 7.0-7.2,115 DEG C of height
Press steam sterilizing 15min.
It is as follows that 5L ferment tank produces the step of l-tyrosine:
(1) seed culture: as seed culture fluid OD600When reaching 10-15, prepare access fermentation medium；
(2) seed liquor: being seeded in fermentation medium by fermented and cultured by the inoculum concentration of 10-15%, and fermentation adds when starting
10g/L xylose is added to induce destination gene expression, cultivation temperature is 37 DEG C, and controlling medium pH by auto-feeding ammonium hydroxide is 7.0-
7.2, by stirring and Ventilation Control dissolved oxygen in 25-30%, defoamed with GPE, when glucose exhausts in culture medium, stream of pulses
The glucose solution for adding 80% controls dissolved oxygen 30% when the l-tyrosine crystal in fermentation liquid is combined together with foam
More than.
Preferred fermentation medium composition are as follows: glucose 10g/L, xylose 10g/L, yeast powder 5g/L, (NH4)2SO45g/L,
KH2PO43g/L, MgSO4·7H2O 2g/L, sodium citrate 2g/L, CaCl2·2H2O 0.015g/L, FeSO4·7H2O 30mg/
L, micro-mixed liquor (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,
H3BO30.125g/L) 1.5mL/L, VB10.5mg/L, VH0.5mg/L, L-phenylalanine 0.5g/L, pH 7.0-7.2,115 DEG C of height
Press steam sterilizing 15min.
The method have the advantages that:
The bacterial strain genetic background of l-tyrosine genetic engineering bacterium provided by the invention is clear, and genetic manipulation is simple and efficient, bacterium
Strain does not carry plasmid, and addition antibiotic is not needed in fermentation process, controls key gene with T7 strong promoter, is lured using xylose
The high efficient expression for leading key gene, reduces production cost, avoids injury of the inducers such as IPTG to cell, zymotechnique
Simply, controllability is strong, and for 24 hours, yield reaches 6.8g/L, 5L ferment tank 30h to shake flask fermentation, and yield is suitable for up to 37.8g/L
Industrialization large-scale production.
Specific embodiment
Below by specific implementation example, the invention will be further described:
A kind of method that the present invention discloses l-tyrosine genetic engineering bacterium and its produces l-tyrosine, the genetic engineering bacterium
It is the method by metabolic engineering, correspondence is carried out to l-tyrosine synthesis related gene in Escherichia coli in genome level and is changed
It makes, blocks competition approach phenylalanine route of synthesis, release the feedback inhibition and transcription negative regulation of key enzyme, and opened using xylose
Mover induction derives from the RNA polymerase of T7 bacteriophage, in conjunction with the high efficient expression of T7 strong promoter system starting key gene.
L-tyrosine 6.8g/L can be accumulated for 24 hours by carrying out shake flask fermentation using the bacterial strain, reachable in 5L fermentation cylinder for fermentation 30h yield
37.8g/L.The genetic engineering bacterium does not carry plasmid, addition antibiotic is not needed in fermentation process, while inducing using xylose
The high efficient expression of key gene, reduces production cost, avoids injury of the inducers such as IPTG to cell, the production L-
The method of tyrosine is simple, controllability is strong, is suitable for industrialization large-scale production.
Embodiment 1: the building of Escherichia coli l-tyrosine engineering strain
The knockout of following gene and integration are all made of Escherichia coli CRISPR/Cas9 gene editing technology.
(1) knockout of pheLA gene
1. using E.coli W3110 (ATCC 27325) genome as template, according to pheLA gene order, respectively in gene
Upstream homology arm primer (pheLA-1, pheLA-2) and downstream homology arm primer (pheLA-3, pheLA-4) are designed in both ends, pass through
The upstream and downstream homology arm of HS enzyme PCR amplification acquisition pheLA gene；
2. using step, 1. the middle amplified fragments obtained pass through HS enzyme over-lap PCR and obtain pheLA gene knockout piece as template
Section, the gene knockout segment are made of pheLA gene upstream and downstream homology arm；
3. containing target by what annealing building pGRB-pheLA was used using pGRB-pheLA-F and pGRB-pheLA-R as primer
The DNA fragmentation of sequence, and changed and go to DH5 αization and turn in competent cell, screening obtains positive transformant, extracts plasmid
pGRB-pheLA；
4. by step 2. and 3. in the pheLA gene knockout segment that obtains and pGRB-pheLA plasmid simultaneously electrotransformation to containing
There is the E.coliW3110 electricity of pREDCas9 to turn in competent cell, screening obtains positive transformant, eliminates plasmid pGRB-
Bacterial strain E.coli TYR01 is obtained after pheLA, pREDCas9.
(2) PxylF-T7RNAP gene integration is to the site lacZ
1. using E.coli W3110 (ATCC 27325) genome as template, according to xylose encoding gene xylF gene order
Design primer PxylF-1 and PxylF-2 obtain xylose promoter PxylF segment by HS enzyme PCR amplification；
2. using E.coli BL21 (DE3) genome as template, according to T7RNAP gene order design primer T7RNAP-1 and
T7RNAP-2 obtains T7RNAP segment by HS enzyme PCR amplification；
3. using E.coli W3110 (ATCC 27325) genome as template, according to lacZ gene sequence, respectively in gene
Upstream homology arm primer (lacZ-1, lacZ-2) and downstream homology arm primer (lacZ-3, lacZ-4) are designed in both ends, pass through HS enzyme
The upstream and downstream homology arm of PCR amplification acquisition lacZ gene；
4. 1., 2. and 3. the middle amplified fragments obtained pass through HS enzyme over-lap PCR and obtain PxylF- as template using step
T7RNAP gene integration segment, the gene integration segment is by under the upstream lacZ homology arm, PxylF-T7RNAP segment and lacZ
Swim homology arm composition；
5. constructing the sequence containing target that pGRB-lacZ is used by annealing using pGRB-lacZ-F and pGRB-lacZ-R as primer
The DNA fragmentation of column, and changed and go to DH5 αization and turn in competent cell, screening obtains positive transformant, extracts plasmid pGRB-
lacZ；
6. 4. and 5. electricity turns simultaneously for the middle PxylF-T7RNAP gene integration segment obtained and pGRB-lacZ plasmid by step
Change to the E.coli TYR01 electricity containing pREDCas9 and turn in competent cell, screening obtains positive transformant, eliminates plasmid
Bacterial strain E.coli TYR02 is obtained after pGRB-lacZ, pREDCas9.
(3) PT7-tyrA (M53I, A354V)-tyrB gene integration is to the site tyrR
1. using E.coli W3110 (ATCC 27325) genome as template, according to tyrA gene order, respectively in gene
TyrA (M53I) mutation upstream homology arm primer (tyrA (M53I) -1, tyrA (M53I) -2) is designed at both ends and tyrA (M53I) is prominent
Become downstream homology arm primer (tyrA (M53I) -3, tyrA (M53I) -4), wherein tyrA (M53I) -2 and tyrA (M53I) -3 is same
When contain mutant nucleotide sequence (ATG sports ATC) and reverse complemental, pass through HS enzyme PCR amplification obtain tyrA (M53I) gene it is upper and lower
Homology arm is swum, then using its upstream and downstream homology arm as template, is obtained by the amplification of Ex.taq enzyme over-lap PCR prominent containing tyrA (M53I)
The segment of change, the mutant fragments are mutated upstream homology arm by tyrA (M53I) and tyrA (M53I) is mutated downstream homology arm group
At.Change goes to DH5 αization and turns in competent cell after mutant fragments are connect with carrier T, and screening obtains positive transformant, extracts
Sequencing determines successfully building T load-tyrA (M53I) after plasmid；
2., according to tyrA gene order, designing tyrA at gene both ends respectively with T load-tyrA (M53I) for template
(A354V) it is mutated upstream homology arm primer (tyrA (M53I) -1, tyrA (A354V) -1) and tyrA (A354V) mutation downstream is same
Source arm primer (tyrA (A354V) -2, tyrA (M53I) -4), wherein tyrA (A354V) -1 and tyrA (A354V) -2 is simultaneously containing prominent
Become sequence (GCA sports GTC) and reverse complemental, the upstream and downstream for obtaining tyrA (M53I) gene by HS enzyme PCR amplification are homologous
Arm, then using its upstream and downstream homology arm as template, obtained by the amplification of Ex.taq enzyme over-lap PCR prominent containing tyrA (M53I, A354V)
The segment of change, the mutant fragments are made of mutation upstream homology arm and mutation downstream homology arm.Mutant fragments and carrier T are connected
Change goes to DH5 αization and turns in competent cell after connecing, and screening obtains positive transformant, and sequencing, which determines, after extraction plasmid successfully constructs T
Load-tyrA (M53I, A354V)；
3. using E.coli W3110 (ATCC 27325) genome as template, according to tyrR gene order, respectively in gene
The upstream tyrR homology arm primer (tyrR-1, tyrR-2) and the downstream tyrR homology arm primer (tyrR-3, tyrR-4) are designed in both ends,
According to tyrB gene order design primer (tyrB-1, tyrB-2), with step 2. in the T load-tyrA (M53I, A354V) that constructs
For template, according to tyrA gene order design primer (tyrA (M53I, A354V) -1, tyrA (M53I, A354V) -2), wherein T7
Promoter sequence designs on tyrR-2 and tyrA (M53I, A354V) -1, and T7 terminator sequence is designed in tyrB-2 and tyrR-3
On, each genetic fragment is obtained by HS enzyme PCR amplification, then using it as template, PT7-tyrA is obtained by HS enzyme over-lap PCR
(M53I, A354V)-tyrB gene integration segment, the gene integration segment by the upstream tyrR homology arm, PT7-tyrA (M53I,
A354V)-tyrB segment and the downstream tyrR homology arm composition；
4. constructing the sequence containing target that PGRB-tyrR is used by annealing using pGRB-tyrR-F and pGRB-tyrR-R as primer
The DNA fragmentation of column, and changed and go to DH5 αization and turn in competent cell, screening obtains positive transformant, extracts plasmid pGRB-
tyrR；
5. by step 3. and 4. in PT7-tyrA (M53I, the A354V)-tyrB gene integration segment that obtains and pGRB-
Electrotransformation to the E.coli TYR02 electricity containing pREDCas9 turns in competent cell tyrR plasmid simultaneously, and screening obtains positive turn
Beggar obtains bacterial strain E.coli TYR03 after eliminating plasmid pGRB-tyrR, pREDCas9.
(4) PT7-aroG (S180F) gene integration is to the site yghX
1. using E.coli W3110 (ATCC 27325) genome as template, according to aroG gene order, respectively in gene
Design aroG (S180F) mutation upstream homology arm primer (aroG (S180F) -1, aroG (S180F) -2) and aroG in both ends
(S180F) be mutated downstream homology arm primer (aroG (S180F) -3, aroG (S180F) -4), wherein aroG (S180F) -2 and
AroG (S180F) -3 contains mutant nucleotide sequence (TCT sports TTT) and reverse complemental simultaneously, is expanded and is obtained by HS enzyme round pcr
The upstream and downstream homology arm of aroG (S180F) gene, then using its upstream and downstream homology arm as template, pass through Ex.taq enzyme over-lap PCR
The segment containing aroG (S180F) mutation is obtained, the mutant fragments are mutated upstream homology arm and aroG by aroG (S180F)
(S180F) mutation downstream homology arm composition.Change goes to DH5 αization and turns in competent cell after mutant fragments are connect with carrier T,
Screening obtains positive transformant, and sequencing determines successfully building T load-aroG (S180F) after extracting plasmid；
2. using E.coli W3110 (ATCC 27325) genome as template, according to yghX gene order, respectively in gene
The upstream yghX homology arm primer (yghX-1, yghX-2) and the downstream yghX homology arm primer (yghX-3, yghX-4) are designed in both ends,
With step 1. in construct T load-aroG (S180F) be template, according to aroG gene order design primer (aroG (S180F) -5,
AroG (S180F) -6), wherein the design of T7 promoter sequence is on yghX-2 and aroG (S180F) -5, the design of T7 terminator sequence
On aroG (S180F) -6 and yghX-3, each genetic fragment is obtained by HS enzyme PCR amplification, then using it as template, pass through HS enzyme
Over-lap PCR obtains PT7-aroG (S180F) gene integration segment, and the gene integration segment is by the upstream yghX homology arm, PT7-
AroG (S180F) and the downstream yghX homology arm composition；
3. constructing the sequence containing target that PGRB-yghX is used by annealing using pGRB-yghX-F and pGRB-yghX-R as primer
The DNA fragmentation of column, and changed and go to DH5 αization and turn in competent cell, screening obtains positive transformant, extracts plasmid pGRB-
yghX；
4. by step 2. and 3. in PT7-aroG (S180F) the gene integration segment that obtains and pGRB-yghX plasmid simultaneously
Electrotransformation to the E.coli TYR03 electricity containing pREDCas9 turns in competent cell, and screening obtains positive transformant, eliminates matter
Bacterial strain E.coli TYR04 is obtained after grain pGRB-yghX, pREDCas9.
Above-mentioned experimentation the primer see the table below:
Embodiment 2
Shake flask fermentation experiment
(1) seed culture: slant strains are seeded in the 500mL round bottom triangular flask that liquid amount is 30mL, nine layers of gauze
Sealing, 37 DEG C, 200rmp culture 10h；
(2) it fermented and cultured: is seeded to by 10% inoculum concentration in the 500mL baffle flask that liquid amount is 30mL, nine layers
Gauze sealing, 37 DEG C, 200rmp culture, addition 10g/L xylose induces destination gene expression when fermentation starts, in fermentation process with
Phenol red makees indicator, and the ammonium hydroxide by adding 25% controls pH in 7.0-7.2, is maintained by the glucose solution for adding 60%
Fermentation carries out.For 24 hours, l-tyrosine yield is 6.8g/L for fermentation.
Fermentation medium composition are as follows: glucose 10g/L, xylose 10g/L, yeast powder 5g/L, (NH4)2SO45g/L,
KH2PO43g/L, MgSO4·7H2O 2g/L, sodium citrate 2g/L, CaCl2·2H2O 0.015g/L, FeSO4·7H2O 30mg/
L, micro-mixed liquor (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,
H3BO30.125g/L) 1.5mL/L, VB10.5mg/L, VH0.5mg/L, L-phenylalanine 0.5g/L, pH 7.0-7.2,115 DEG C of height
Press steam sterilizing 15min.
Embodiment 3
The specific method is as follows for 5L ferment tank:
(1) seed culture: aseptically, appropriate amounts of sterilized water being poured into inclined-plane, thallus is suspended with oese, so
Bacterial suspension inoculation is cultivated in seed culture medium by the way of flame inoculation afterwards.Cultivation temperature is 37 DEG C, initial logical
Tolerance is 2L/min, and initial speed of agitator is 200r/min, and controlling medium pH by the ammonium hydroxide of auto-feeding 25% is 7.0-
7.2, it when dissolved oxygen is lower than 25%, by stirring and Ventilation Control dissolved oxygen in 25-30%, is defoamed with GPE, every 2h test sample, note
Data are recorded, OD is worked as600When reaching 10-15, prepare access fermentation medium.
(2) seed liquor: being seeded in fermentation medium by fermented and cultured by 10% inoculum concentration, and fermentation is added when starting
10g/L xylose induces destination gene expression, and cultivation temperature is 37 DEG C, controls medium pH by the ammonium hydroxide of auto-feeding 25% and is
7.0-7.2 is defoamed by stirring and Ventilation Control dissolved oxygen in 25-30% with GPE, when glucose exhausts in culture medium,
Stream of having a rest adds 80% glucose solution, and controlling glucose remaining sugar concentration in fermentation liquid is 0.05-5g/L, when the L- junket in fermentation liquid
When propylhomoserin crystal and foam are combined together, dissolved oxygen is controlled 30% or more.Ferment 30h, and l-tyrosine yield is 37.8g/L.
Intermittent flow, which adds, to be referred to: being adjusted according to glucose remaining sugar concentration in fermentation liquid and is mended sugared pulse ratio (18-30:1), had been provided
Remaining sugar concentration specific value.
Fermentation medium composition are as follows: glucose 10g/L, xylose 10g/L, yeast powder 5g/L, (NH4)2SO45g/L,
KH2PO43g/L, MgSO4·7H2O 2g/L, sodium citrate 2g/L, CaCl2·2H2O 0.015g/L, FeSO4·7H2O 30mg/
L, micro-mixed liquor (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,
H3BO30.125g/L) 1.5mL/L, VB10.5mg/L, VH0.5mg/L, L-phenylalanine 0.5g/L, pH 7.0-7.2,115 DEG C of height
Press steam sterilizing 15min.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>University Of Science and Technology Of Tianjin
<120>a kind of l-tyrosine genetic engineering bacterium and its method for producing l-tyrosine
<160> 44
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> pheLA-1(Unknown)
<400> 1
caccaagacg ggaagacaag a 21
<210> 2
<211> 32
<212> DNA
<213> pheLA-2(Unknown)
<400> 2
gattcaagat tcccgtactg ttttcacgct gt 32
<210> 3
<211> 32
<212> DNA
<213> pheLA-3(Unknown)
<400> 3
acagtacggg aatcttgaat cagcggaaat gc 32
<210> 4
<211> 21
<212> DNA
<213> pheLA-4(Unknown)
<400> 4
cgagcacgat cagaatatgg c 21
<210> 5
<211> 56
<212> DNA
<213> pGRB-pheLA-F(Unknown)
<400> 5
agtcctaggt ataatactag tgcgagagaa aatcagcgcg cgttttagag ctagaa 56
<210> 6
<211> 56
<212> DNA
<213> pGRB-pheLA-R(Unknown)
<400> 6
ttctagctct aaaacgcgcg ctgattttct ctcgcactag tattatacct aggact 56
<210> 7
<211> 37
<212> DNA
<213> PxylF-1(Unknown)
<400> 7
gttctgtctc ggcggagata attcacaagt gtgcgct 37
<210> 8
<211> 41
<212> DNA
<213> PxylF-2(Unknown)
<400> 8
ttaatcgtgt tcatttagtg cctcttccag ttagtaaatc c 41
<210> 9
<211> 42
<212> DNA
<213> T7RNAP-1(Unknown)
<400> 9
actggaagag gcactaaatg aacacgatta acatcgctaa ga 42
<210> 10
<211> 33
<212> DNA
<213> T7RNAP-2(Unknown)
<400> 10
cgcaactcgc cccggcacag tatcaaggta ttt 33
<210> 11
<211> 20
<212> DNA
<213> lacZ-1(Unknown)
<400> 11
acaacaactg gcgggcaaac 20
<210> 12
<211> 36
<212> DNA
<213> lacZ-2(Unknown)
<400> 12
ttgtgaatta tctccgccga gacagaactt aatggg 36
<210> 13
<211> 32
<212> DNA
<213> lacZ-3(Unknown)
<400> 13
atactgtgcc ggggcgagtt gcgtgactac ct 32
<210> 14
<211> 20
<212> DNA
<213> lacZ-4(Unknown)
<400> 14
cacagcggat ggttcggata 20
<210> 15
<211> 55
<212> DNA
<213> pGRB-lacZ-F(Unknown)
<400> 15
agtcctaggt ataatactag tcgtcgtgac tgggaaaacc cgttttagag ctaga 55
<210> 16
<211> 56
<212> DNA
<213> pGRB-lacZ-R(Unknown)
<400> 16
ttctagctct aaaacgggtt ttcccagtca cgacgactag tattatacct aggact 56
<210> 17
<211> 22
<212> DNA
<213> aroG(S180F)-1(Unknown)
<400> 17
caccgctggc aaggcttaga gt 22
<210> 18
<211> 30
<212> DNA
<213> aroG(S180F)-2(Unknown)
<400> 18
gaagccgacc ggacaaaaaa gccctgatgc 30
<210> 19
<211> 30
<212> DNA
<213> aroG(S180F)-3(Unknown)
<400> 19
gcatcagggc ttttttgtcc ggtcggcttc 30
<210> 20
<211> 22
<212> DNA
<213> aroG(S180F)-4(Unknown)
<400> 20
caaaccaggg taaagcgaag ta 22
<210> 21
<211> 21
<212> DNA
<213> yghX-1(Unknown)
<400> 21
gcgcaacgta gaacaggaat t 21
<210> 22
<211> 66
<212> DNA
<213> yghX-2(Unknown)
<400> 22
agttaaacaa aattatttct agaccctata gtgagtcgta ttagattgaa gcgcctttac 60
tactcc 66
<210> 23
<211> 62
<212> DNA
<213> yghX-3(Unknown)
<400> 23
cttggggcct ctaaacgggt cttgaggggt tttttggtca tagtaatcca gcaactcttg 60
tg 62
<210> 24
<211> 22
<212> DNA
<213> yghX-4(Unknown)
<400> 24
gagcaggtat ttacgtgaac cg 22
<210> 25
<211> 68
<212> DNA
<213> aroG(S180F)-5(Unknown)
<400> 25
ggtctagaaa taattttgtt taactttaag aaggagatat accatgaatt atcagaacga 60
cgatttac 68
<210> 26
<211> 62
<212> DNA
<213> aroG(S180F)-6(Unknown)
<400> 26
tcaagacccg tttagaggcc ccaaggggtt atgctagacg tcattcgttt aaaatgagaa 60
ag 62
<210> 27
<211> 55
<212> DNA
<213> pGRB-yghX-F(Unknown)
<400> 27
agtcctaggt ataatactag tggtgcctga cgaccataaa agttttagag ctaga 55
<210> 28
<211> 56
<212> DNA
<213> pGRB-yghX-R(Unknown)
<400> 28
ttctagctct aaaactttta tggtcgtcag gcaccactag tattatacct aggact 56
<210> 29
<211> 20
<212> DNA
<213> tyrA(M53I)-1(Unknown)
<400> 29
ccacgagggc aatcagtctt 20
<210> 30
<211> 20
<212> DNA
<213> tyrA(M53I)-2(Unknown)
<400> 30
aggccaagat agatgcctcg 20
<210> 31
<211> 20
<212> DNA
<213> tyrA(M53I)-3(Unknown)
<400> 31
cgaggcatct atcttggcct 20
<210> 32
<211> 20
<212> DNA
<213> tyrA(M53I)-4(Unknown)
<400> 32
agcgacaaaa cttcacccga 20
<210> 33
<211> 19
<212> DNA
<213> tyrA(A354V)-1(Unknown)
<400> 33
aaacgctgga cgtaatcgc 19
<210> 34
<211> 19
<212> DNA
<213> tyrA(A354V)-2(Unknown)
<400> 34
gcgattacgt ccagcgttt 19
<210> 35
<211> 21
<212> DNA
<213> tyrR-1(Unknown)
<400> 35
agccgtttgc gtctgtttaa g 21
<210> 36
<211> 60
<212> DNA
<213> tyrR-2(Unknown)
<400> 36
taaacaaaat tatttctaga ccctatagtg agtcgtatta gggcacttcg gcgtaaagat 60
<210> 37
<211> 56
<212> DNA
<213> tyrR-3(Unknown)
<400> 37
tggggcctct aaacgggtct tgaggggttt tttggttgcg ggaatatggt ctgagt 56
<210> 38
<211> 22
<212> DNA
<213> tyrR-4(Unknown)
<400> 38
agtgctgaac attttcccga gt 22
<210> 39
<211> 38
<212> DNA
<213> tyrB-1(Unknown)
<400> 39
taaaaggaga tatacccatc gcgtgtttca aaaagttg 38
<210> 40
<211> 62
<212> DNA
<213> tyrB-2(Unknown)
<400> 40
agacccgttt agaggcccca aggggttatg ctagaagctt aacaataaaa gaactaacaa 60
cg 62
<210> 41
<211> 61
<212> DNA
<213> tyrA(M53I, A354V)-1(Unknown)
<400> 41
tctagaaata attttgttta actttaagaa ggagatatac catggttgct gaattgaccg 60
c 61
<210> 42
<211> 38
<212> DNA
<213> tyrA(M53I, A354V)-2(Unknown)
<400> 42
gatgggtata tctcctttta ctggcgattg tcattcgc 38
<210> 43
<211> 56
<212> DNA
<213> pGRB-tyrR-F(Unknown)
<400> 43
agtcctaggt ataatactag tctcgatcta ctcgtgctaa ggttttagag ctagaa 56
<210> 44
<211> 56
<212> DNA
<213> pGRB-tyrR-R(Unknown)
<400> 44
ttctagctct aaaaccttag cacgagtaga tcgagactag tattatacct aggact 56
Claims (7)
1. a kind of l-tyrosine genetic engineering bacterium, it is characterised in that: the genetic engineering bacterium has knocked out L-phenylalanine synzyme
The T7 phage rna polymerase that xylose promoter PxylF is controlled is integrated into lacZ by gene pheA and its controlling gene pheL
TyrA (M53I, the A354V)-tyrB controlled by T7 promoter is integrated into the site tyrR, the aroG of T7 promoter control by point
(S180F) it is integrated into yghX pseudogene site.
2. a kind of l-tyrosine genetic engineering bacterium as described in claim 1, which is characterized in that the genetic engineering bacterium with
E.coli W3110 is starting strain.
3. the method for constructing l-tyrosine genetic engineering bacterium described in claim 1, it is characterised in that: construction step is as follows:
(1) Escherichia coli CRISPR/Cas9 gene editing technology is used, using E.coli W3110 as starting strain, knocks out pheLA
Gene；
(2) junction fragment of xylose promoter PxylF and t7 rna polymerase T7RNAP are constructed, and is integrated into the site lacZ；
(3) junction fragment of PT7 promoter and tyrA (M53I, A354V)-tyrB is constructed, and is integrated into the site tyrR.
(4) junction fragment of PT7 promoter and aroG (S180F) is constructed, and is integrated into yghX pseudogene site.
4. using the method for the production l-tyrosine of l-tyrosine genetic engineering bacterium described in claim 1, it is characterised in that: shaking flask hair
It is as follows that ferment produces the step of l-tyrosine:
Fermented and cultured is carried out by the inoculum concentration of 10-15%, addition 10g/L xylose induces destination gene expression, hair when fermentation starts
Indicator is made with phenol red during ferment, controls pH in 7.0-7.2 by adding ammonium hydroxide, it is molten that chignon adds glucose in the process
Liquid.
5. a kind of method of l-tyrosine genetic engineering bacterium production l-tyrosine as described in claim 1, which is characterized in that 5L
It is as follows that ferment tank produces the step of l-tyrosine:
(1) seed culture: as seed culture fluid OD600When reaching 10-15, prepare access fermentation medium；
(2) seed liquor: being seeded in fermentation medium by fermented and cultured by the inoculum concentration of 10-15%, and fermentation is added when starting
10g/L xylose induces destination gene expression, and cultivation temperature is 37 DEG C, and controlling medium pH by auto-feeding ammonium hydroxide is 7.0-
7.2, by stirring and Ventilation Control dissolved oxygen in 25-30%, defoamed with GPE, when glucose exhausts in culture medium, intermittent flow
The glucose solution for adding 80% controls dissolved oxygen 30% when the l-tyrosine crystal in fermentation liquid is combined together with foam
More than.
6. a kind of method of l-tyrosine genetic engineering bacterium production l-tyrosine, feature exist as described in claim 4 or 5
In: the fermentation medium is glucose 10g/L, xylose 10g/L, yeast powder 5g/L, (NH4)2SO45g/L, KH2PO43g/L,
MgSO4·7H2O 2g/L, sodium citrate 2g/L, CaCl2·2H2O 0.015g/L, FeSO4·7H2O 30mg/L, microelement
Mixed liquor 1.5mL/L, VB10.5mg/L, VH0.5mg/L, L-phenylalanine 0.5g/L, pH 7.0-7.2,115 DEG C of high steams
Sterilize 15min.
7. the method for l-tyrosine genetic engineering bacterium production l-tyrosine as claimed in claim 6, it is characterised in that: micro member
Plain mixed liquor includes: 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。
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|---|---|---|---|
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| CN110551670A (en) * | 2019-09-19 | 2019-12-10 | 天津科技大学 | Genetically engineered bacterium for producing L-leucine and application thereof |
| 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 |
| CN111471636A (en) * | 2020-04-16 | 2020-07-31 | 旖肽(上海)生物科技有限公司 | Genetically engineered bacterium for expressing human epidermal growth factor and application thereof |
| WO2021060337A1 (en) | 2019-09-25 | 2021-04-01 | Ajinomoto Co., Inc. | Method for producing 2-methyl-butyric acid by bacterial fermentation |
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Application publication date: 20190125 |