CN104745500B - One plant production L phenylalanines bacterial strain and its application - Google Patents
One plant production L phenylalanines bacterial strain and its application Download PDFInfo
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Abstract
The invention discloses one plant of bacterial strain for producing L phenylalanines and its application.One plant of recombination bacillus coli MD1078 disclosed by the invention（Escherichia coli MD1078）, its deposit number is CCTCC NO:M2013509.The bacterial strain is in 100L automatic fermenters, and 36 DEG C, dissolved oxygen 35% 50%, fermentation time are that L phenylalanine yield reaches 86.5g/L under conditions of 48 hours.The ability of Escherichia coli MD1078 production L phenylalanines is high, is had broad application prospects in the actual production of L phenylalanines.
Description
Technical field
The present invention relates to one plant of bacterial strain for producing L-phenylalanine and its application.
Background technology
L-phenylalanine（L-phenylalanine）It is that human body and animal can not in vivo voluntarily for white crystalline powder
One of necessary amino acid of synthesis, is widely used in the fields such as food, feed, medicine and cosmetics.L-phenylalanine is used as conjunction
Into Aspartame（Aspartame）One of two kinds of raw materials, in the dipeptide sweetener Aspartame for preparing low in calories, high sugariness
Application it is increasingly extensive, the market demand of L-phenylalanine increases sharply.Other L-phenylalanine be also antineoplastic and
The required raw material of amino acid transfusion preparation, with its continually developing in field of medicaments, demand is also continuously increased.Therefore, it is right
The research of L-phenylalanine biosynthesis pathway, the improvement of L-phenylalanine producing strains and more next to the research of its industrialized production
More it is valued by people.
The production method of domestic and international L-phenylalanine mainly has hydrolysis extraction process, chemical synthesis, enzyme process and fermentation method etc..
Because L-phenylalanine content is relatively low in native protein, hydrolysis extraction process is rarely employed；The complex process of chemical synthesis, cost
It is higher, substantially replaced abroad by enzyme process and fermentation method.But because the price of substrate and enzyme is higher and limited source, enzyme process should
With also restrained；Because microorganism direct fermentation can utilize raw material cheap and easy to get, it can at normal temperatures and pressures carry out again, be
The main stream approach of L-phenylalanine is produced both at home and abroad at present, with larger competitive advantage.But it is due to that L-phenylalanine etc. is whole
Product has strong feedback inhibition to the key enzyme on the aromatic amino acid metabolic pathway of synthesizing in wild strain body,
Prevent its cell from largely accumulating phenylalanine, therefore to carry out real attenuation production, just need acquisition production L-phenylalanine badly
The high bacterial strain of ability.
When so-called fallibility round pcr refers to amplifying target genes in vitro, using the low fidelity of Taq enzyme, adjust simultaneously
Whole reaction condition, such as improves magnesium ion concentration, the proportional concentration method for adding manganese ion, changing dNTP, with certain frequency
Base mispairing is randomly incorporated into target gene, causes the random mutation of target gene to constitute mutation library, then selects or screen
Go out the mutant of needs.
The content of the invention
It is an object of the invention to provide one plant of bacterial strain for producing L-phenylalanine and its application.
One plant of recombination bacillus coli MD1078 that the present invention is provided（Escherichia coli MD1078）, its preservation volume
Number be CCTCC NO:M 2013509.
Deposit number is CCTCC NO:Application of M 2013509 Escherichia coli in production L-phenylalanine is fallen within
Protection scope of the present invention.
A kind of method for producing L-phenylalanine falls within protection scope of the present invention, and this method comprises the following steps：Will
Deposit number is CCTCC NO:M2013509 Escherichia coli fermentation culture, obtains L-phenylalanine.
In the above method, composition of the fermentation medium used in the fermented and cultured per 100ml is as follows：
Glucose 2g, ammonium sulfate 2g, sodium citrate 0.05g, magnesium sulfate 0.08g, potassium dihydrogen phosphate 0.1g, dipotassium hydrogen phosphate
0.1g, dusty yeast 0.1g, sodium glutamate 0.05g, MnCl20.2mg, niacin 1mg, CoCl20.01mg, ZnSO40.1mg,
CaCl20.5mg, VB11mg, MnCl20.2mg, tyrosine 3mg, surplus is water.
In any of the above-described described method, the condition of the fermentation is 36 DEG C, dissolved oxygen 35%-50%, when in fermentation system
Glucose content starts to add glucose when being less than 15g/L, the content of glucose is maintained between 10g/L-15g/L.
In any of the above-described described method, the time of the fermentation is 48 hours.
In any of the above-described described method, the bacterial strain is to access fermentation medium by seed liquor.
In any of the above-described described method, the composition for preparing the every 100ml of culture medium of the seed liquor is as follows：Glucose
3g, ammonium sulfate 1g, magnesium sulfate 0.08g, potassium dihydrogen phosphate 0.1g, dipotassium hydrogen phosphate 0.1g, dusty yeast 0.2g, sodium glutamate
0.08g, MnCl20.25mg, CoCl20.005mg, ZnSO40.15mg, CaCl20.5mg, VB11mg, MnCl20.2mg, tyrosine
3mg, surplus is water.
In any of the above-described described method, condition of culture when prepared by the seed liquor is 36 DEG C, dissolved oxygen is 40%-60%,
The content of glucose is 30g/L.
In any of the above-described described method, the incubation time of the seed liquor is 12 hours.
It is demonstrated experimentally that the deposit number provided using the present invention is CCTCC NO:M 2013509 Escherichia coli production L-
Phenylalanine, the yield of L-phenylalanine is up to 86.5g/L, it was demonstrated that the ability of bacterial strain production L-phenylalanine of the present invention is very high,
Had broad application prospects in the actual production of L-phenylalanine.
Brief description of the drawings
Fig. 1 is pBV220-aroG-pheA plasmids.
Fig. 2 is the HPLC spectrograms of amino acid standard liquid.
Fig. 3 is the HPLC spectrograms of the dilution of Escherichia coli MD1078 fermented liquid supernatants.
The HPLC spectrograms of the dilution of the fermented liquid supernatant of Fig. 4 Escherichia coli 10245.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
PMD-18T is purchased from precious bioengineering（Dalian）Co., Ltd, catalog number is D101A.
PBV220 is purchased from Beijing Ding Guo bio tech ltd.
Escherichia coli（Escherichia coli）Purchased from Chinese industrial Microbiological Culture Collection administrative center
（www.china-cicc.org）, deposit number is 10245, abbreviation Escherichia coli 10245.
Fermentation medium：50g/L glucose, 10g/L yeast extracts, 3g/L Na2HPO4、1g/L KH2PO4、12g/L
NH4Cl、1g/L MgSO4、0.3g/L CaCl2, 6g/L sodium citrates, 0.6g/L sodium glutamates, 2g/L peptones, 20mg/LVB1,
Surplus is water.
L-phenylalanine is full of pool purchased from Beijing and taken in the fresh Chemical Engineering Technology research institute, and production number is GF2212.
Prepared as follows per 100ml fermentation tank seed culture mediums：
Glucose 3g, ammonium sulfate 1g, magnesium sulfate 0.08g, potassium dihydrogen phosphate 0.1g, dipotassium hydrogen phosphate 0.1g, dusty yeast
0.2g, sodium glutamate 0.08g, MnCl20.25mg, CoCl20.005mg, ZnSO40.15mg, CaCl20.5mg, VB11mg,
MnCl20.2mg, tyrosine 3mg, add water and are settled to 100ml, sterilizing.
Prepared as follows per 100ml ferment tank culture mediums：
Glucose 2g, ammonium sulfate 2g, sodium citrate 0.05g, magnesium sulfate 0.08g, potassium dihydrogen phosphate 0.1g, dipotassium hydrogen phosphate
0.1g, dusty yeast 0.1g, sodium glutamate 0.05g, MnCl20.2mg, niacin 1mg, CoCl20.01mg, ZnSO40.1mg,
CaCl20.5mg, VB11mg, MnCl20.2mg, tyrosine 3mg, add water and are settled to 100ml, sterilizing.
Amino acid standard liquid is purchased from Tianjin Bonaaijieer Technology Co., Ltd.
The preparation of embodiment 1, the bacterial strain of production L-phenylalanine
First, recombinant plasmid pBV220-aroG-pheA structure
The genomic DNA of Escherichia coli 10245 is extracted, using it as template, is primer with primer aroG-f and aroG-r, enters
Performing PCR is expanded, and obtains aroG genes.
The genomic DNA of Escherichia coli 10245 is extracted, using it as template, is primer with primer pheA-f and pheA-r, enters
Performing PCR is expanded, and obtains pheA genes.
EcoR I and the double digestion aroG genes of Kpn I, obtain genetic fragment；The EcoR I and double digestion pMD-18T of Kpn I, is obtained
Carrier large fragment；Genetic fragment is connected with carrier large fragment, recombinant plasmid is obtained, pMD-18T-aroG is named as.
Kpn I and the double digestion pheA genes of BamH I, obtain genetic fragment；The Kpn I and double digestion pMD-18T of BamH I, is obtained
Carrier large fragment；Genetic fragment is connected with carrier large fragment, recombinant plasmid is obtained, pMD-18T-pheA is named as.
aroG-f:5’-CGGAATTCATGAATTATCAGAACGACGAT-3’
aroG-r:5’-GGGGTACCACCCGCGACGCGCTTTTACTG-3’
pheA-f:5’-GGGGTACCATGACATCGGAAAACCCGTTAC-3’
pheA-r:5’-CGGGATCCTCAGGTTGGATCAACAGGCAC-3’
（Sequence shown in underscore is digestion recognition site）
With Kpn I and the double digestion pMD-18T-aroG of BamH I, carrier large fragment is reclaimed；With Kpn I and the double digestions of BamH I
PMD-18T-pheA, reclaims pheA genetic fragments；Carrier large fragment and pheA genetic fragments are connected, recombinant plasmid is obtained, ordered
Entitled pMD-aroG-pheA.PMD-aroG-pheA is subjected to sequence verification, as a result correctly.
Plasmid pMD-aroG-pheA is subjected to double digestion with EcoR I and BamH I, aroG-pheA genetic fragments are reclaimed；Will
Plasmid pBV220 carries out double digestion with EcoR I and BamH I, reclaims carrier large fragment；By carrier large fragment and aroG-pheA genes
Fragment is connected, and is obtained recombinant plasmid, is named as pBV220-aroG-pheA, as shown in figure 1, sequence verification is correct.
2nd, recombinant plasmid pBV220-aroG-pheA promutation
（One）Fallibility PCR is mutated
With plasmid pBV220-aroG-pheA templates, by improving magnesium ion concentration and changing dNTP concentration, fallibility is carried out
PCR, makes gene aroG-pheA undergo mutation, and PCR primer is gene aroG-pheA mutation library.
Sense primer aroG-f:5’-CGGAATTCATGAATTATCAGAACGACGAT-3’
Anti-sense primer pheA-r:5’-CGGGATCCTCAGGTTGGATCAACAGGCAC-3’；
（Sequence shown in underscore is digestion recognition site）
Error-prone PCR systems（100μl）：
DATP, dCTP, dTTP, dGTP concentration are 100mM/mL, MgCl2Concentration be 25mM/mL, upstream and downstream primer
Concentration is 10 μm of ol/L, and the concentration of DNA template is 70ng/ μ L.
PCR programs are：94 DEG C of pre-degeneration 4min；94 DEG C of denaturation 1min, 56 DEG C of annealing 1min, 72 DEG C of extension 1min40s, instead
Answer 40 circulations；Last 72 DEG C of extensions 10min, takes 5 μ lPCR products to carry out 1% agarose gel electrophoresis inspection, -20 DEG C of preservations
It is standby.
（Two）DNA reorganizes
The template reorganized using above-mentioned fallibility PCR amplified production as DNA, using pfu as archaeal dna polymerase, by it is following operate into
Performing PCR is expanded, and obtained pcr amplification product is the aroG-pheA mutant libraries after DNA reorganization：
Sense primer aroG-f:5’-CGGAATTCATGAATTATCAGAACGACGAT-3’
Anti-sense primer pheA-r:5’-CGGGATCCTCAGGTTGGATCAACAGGCAC-3’；
（Sequence shown in underscore is digestion recognition site）
PCR programs：
（1）94 DEG C, 1min53 DEG C, 1min72 DEG C, 20s, 13 circulations
（2）94 DEG C, 1min55 DEG C, 1min15s72 DEG C, 20s, 25 circulations
（3）94 DEG C, 1min54 DEG C, 1min35s72 DEG C, 20s, 75 circulations
72 DEG C of insulation 10min make extension complete.
PCR system is：
DNTP concentration is 100mM/mL, and upstream and downstream primer concentration is 10 μm of ol/L, fallibility PCR primer concentration 60ng/ μ
L。
（Three）The structure of mutant expression library 1
After aroG-pheA mutant library restriction enzymes EcoR I and BamH I after DNA is reorganized are double digested,
Be attached with the pBV220 plasmids of identical endonuclease digestion, by connection product Transformed E .coli JM109, carry out screening and
Expand culture, obtain the E.coli JM109 bacterium storehouse of the mutant containing pBV220-aroG-pheA.
Plasmid is extracted from E.coli JM109 bacterium storehouse, plasmid is converted into Escherichia coli 10245, obtained containing pBV220-
The bacterium storehouse of Escherichia coli 10245 of aroG-pheA mutant, is denoted as mutant expression library 1.
（Four）L-phenylalanine Producing Strain is screened from storehouse 1
The monoclonal of picking mutant expression library 1 to every 150 μ L of hole g/mL concentration of μ containing Amp100 fermentation medium 96
In orifice plate, 37 DEG C, 150rpm culture 48h, 3000rpm centrifugations take supernatant, to be measured.
Three wave bands survey the content of L-phenylalanine in acid system detection fermented supernatant fluid：
1st, the 0.1g/100ml L-phenylalanine aqueous solution is prepared to dilute as standard items, then with 0.1mol/L hydrochloric acid solutions
After 1000 times, using 0.1mol/L hydrochloric acid solutions as blank, absorbance A is surveyed206, A208, A222。
2nd, fermentation broth sample supernatant is with after n times of distilled water diluting（L-phenylalanine should be in the Sample supernatants finally measured
0.1g/100ml or so, each absorbance is defined between 0.2-0.7）, 4000r/min centrifugations 15 minutes.
3rd, take each supernatant to dilute 1000 times with 0.1mol/L hydrochloric acid solutions again, using 0.1mol/L hydrochloric acid as blank, determine
Survey absorbance A206, A208, A222。
4th, the concentration of phenylalanine in Sample supernatants is calculated according to formula 1（g/100ml）.
△ samples=A206 samples+0.2727A208 samples-0.9682A222 samples
△ marks=A206 marks+0.2727A208 marks-0.9682A222 marks
Extension rate：1000
Standard concentration：0.1g/100mL
4 plants of orifice plate L-phenylalanine yield of screening acquisition reach that more than 0.755g/100mL's is excellent from the strain of nearly ten thousand plant mutant
Good mutant strain, is denoted as bacterial strain 1- bacterial strains 4.
Same method detection control bacterium Escherichia coli 10245 are only 0.25g/ in the L-phenylalanine yield of orifice plate
100mL。
3rd, recombinant plasmid pBV220-aroG-pheA second of DNA reorganization
（One）Bacterial strain 1- bacterial strains 4 mix the preparation of plasmid
The monoclonal of picking bacterial strain 1- bacterial strains 4 is trained overnight in the LB culture mediums of 3mL μ containing Amp100 g/mL concentration respectively
Support, then extract the DNA of bacterial strain 1- bacterial strains 4, the template that 4 kinds of plasmid mixed in equal amounts are reorganized as DNA.
（Two）DNA reorganizes
The template reorganized using the plasmid of above-mentioned mixed in equal amounts as the second round DNA, using pfu as archaeal dna polymerase, by following
Operate into performing PCR amplification, obtain pcr amplification product：
Sense primer aroG-f:5’-CGGAATTCATGAATTATCAGAACGACGAT-3’
Anti-sense primer pheA-r:5’-CGGGATCCTCAGGTTGGATCAACAGGCAC-3’；
PCR programs：
（1）94 DEG C, 1min53 DEG C, 1min72 DEG C, 20s, 13 circulations
（2）94 DEG C, 1min55 DEG C, 1min15s72 DEG C, 20s, 25 circulations
（3）94 DEG C, 1min54 DEG C, 1min35s72 DEG C, 20s, 75 circulations
72 DEG C of insulation 10min make extension complete.
PCR system is：
DNTP concentration is 100mM/mL, and upstream and downstream primer concentration is 10 μm of ol/L, mixing plasmid template concentration 75ng/
μL。
（Three）The structure of mutant expression library 2
By pcr amplification product with restriction enzyme EcoR I and BamH I it is double digested after, disappear with identical restriction endonuclease
The pBV220 plasmids of change are attached, by connection product Transformed E .coli JM109, are screened and expand culture, are contained
The E.coli JM109 bacterium storehouse of pBV220-aroG-pheA mutant.
Plasmid is extracted from E.coli JM109 bacterium storehouse, plasmid is converted to Escherichia coli 10245 respectively, contained
The bacterium storehouse of Escherichia coli 10245 of pBV220-aroG-pheA mutant, is denoted as mutant expression library 2.
（Four）L-phenylalanine Producing Strain is screened from storehouse 2
The monoclonal of picking mutant expression library 2 to every 150 μ L of hole g/mL concentration of μ containing Amp100 fermentation medium 96
In orifice plate, 37 DEG C, 150rpm culture 48h, 3000rpm centrifugations take supernatant, to be measured.
Three wave bands survey the content of L-phenylalanine in acid system detection fermented supernatant fluid, and method is with step 2（Four）.
Screening obtains the bacterial strain that one plant of orifice plate L-phenylalanine yield reaches 0.985g/100mL from nearly ten thousand mutant strains, will
It is named as Escherichia coli MD1078.The yield of the orifice plate L-phenylalanine of remaining bacterial strain is typically arrived in 0.38g/100mL
Between 0.85g/100mL.
Escherichia coli MD1078（Escherichia coli MD1078）Chinese allusion quotation is preserved on October 25th, 2013
Type culture collection（Abbreviation CCTCC；Address：Chinese Wuhan Wuhan Universitys；Postcode：430072）, deposit number is
CCTCC NO:M 2013509。
Sour (L-phenylalanine) ability of the production of embodiment 2, bacterial strain MD1078 and Escherichia coli 10245
Escherichia coli MD1078 and the 50L fermentation tank acid producing abilities of Escherichia coli 10245
First, seed culture（Using 30L automatic fermenters, seed culture medium liquid amount is 20L）：By Escherichia coli
MD1078 and Escherichia coli 10245 are inoculated in seed culture medium respectively, and 30L seed fermentation tanks control parameter is 36 DEG C, dissolved oxygen
（DO）For 40%-60%, concentration of glucose is 30g/L in seed culture medium, and incubation time is 12 hours, obtains seed liquor（Seed
Liquid OD610=30）.
2nd, fermented and cultured（Using 100L automatic fermenters, fermentation liquid culture medium liquid amount is 50L）：Sent out using 100L
Fermentation tank is fermented.Seed liquor is inoculated in fermentation medium by 25% volume, fermentation tank control parameter is 36 DEG C, dissolved oxygen
（DO）When glucose content in 35%-50%, fermentation system is less than 15g/L, 700g/L glucose is added automatically by fermentation tank
The aqueous solution so that the glucose content of fermentation system is maintained between 10g/L-15g/L, fermentation time is 48 hours.
After Escherichia coli MD1078 and 48 hours zymotic fluid 15mL4500rpm of Escherichia coli 10245 centrifugations, it is taken to send out
Ferment supernatant is diluted with water to progress liquid phase detection after 200 times of volumes.
3rd, liquid phase detects the content of L-phenylalanine in fermented liquid supernatant
L-phenylalanine（Phe is denoted as below）The assay method of concentration often uses assay method, i.e., efficient liquid using amino acid
Phase chromatogram（HPLC）Derivatization Method（Rapid,accurate,senstitive,and reprodicobLe HPLC
anaLysis of amino acida.Henderson,J.W,Ricker,R.D.,BidLingmeyer,B.A.,Woodward,
C.,AgiLent TechnoLogier,ΜSA,2000）.
L-phenylalanine concentration in sample solution（μg/mL）=f1/f2×C；
The peak area of L-phenylalanine/interior target peak area in f1=sample solution；
The peak area of L-phenylalanine/interior target peak area in f2=amino acid standard liquid；
L-phenylalanine in C=amino acid standard liquid（μg/mL）.
Chromatographic column：AgeLa VenusiL AA posts, (4.6*250mm, 5 μm)；
Column temperature：40℃；
Detection wavelength：254nm；
Mobile phase A：15.2g anhydrous sodium acetates are taken, add water 1850mL, pH to 6.5, Ran Houjia is adjusted with glacial acetic acid after dissolving
140mL acetonitriles, are mixed, with 0.45 μm of membrane filtration；
Mobile phase B：Acetonitrile and water are according to volume ratio 4:1 prepares；
Flow velocity：1mL/min；
Nor-leucine inner mark solution：Take nor-leucine（Nle）10mg, plus 10mL0.1moL/L aqueous hydrochloric acid solutions, dissolving；
Concentration of phenylalanine is 2.5 μm of oL/mL in amino acid standard liquid；
The derivative of amino acid standard liquid and sample solution, is carried out as follows：
（1）Amino acid standard liquid and analyte sample fluid are measured respectively（The Escherichia coli MD1078 and large intestine bar of step 2
The dilution of the fermented supernatant fluid of bacterium 10245）Each 200 μ L, are respectively placed in 1.5mL centrifuge tubes；
（2）The μ L of nor-leucine inner mark solution 20 are added in each centrifuge tube；
（3）100 μ L triethylamine acetonitrile solutions are added in each centrifuge tube（8.6mL acetonitriles are added in 1.4mL triethylamines, are mixed
It is even）With 100 μ L phenyl isothiocyanate acetonitrile solutions（25 μ L phenyl isothiocyanates add 2mL acetonitriles, mix）, mix, room temperature is put
Put 1 hour；
（4）400 μ L n-hexanes are added in each centrifuge tube, 10min is placed after shaking；
（5）Remove a layer solution, the phenylalanine after as deriving, referred to as PTC- phenylalanines, with 0.45 μm of pin type filter
Filtering；
（6）The μ L of filtrate 200, plus the dilution of 800 μ L water are taken, is shaken up, the μ L of sample introduction 10.
Gradient elution process：0-2min eluent is mobile phase A；2-14min eluent is by mobile phase A and stream
Dynamic phase B compositions, volumn concentration of the mobile phase A in eluent is by 100% linear decline to 93%；14-29min elution
Liquid is made up of mobile phase A and Mobile phase B, and volumn concentration of the mobile phase A in eluent is by 93% linear decline to 70%；The
29-32min eluent is made up of mobile phase A and Mobile phase B, and volumn concentration of the mobile phase A in eluent is by 70% line
Property drops to 50%；32-33min eluent is made up of mobile phase A and Mobile phase B, volume of the mobile phase A in eluent
Percentage composition is by 50% linear rise to 100%；33-39min eluent is mobile phase A；39-45min eluent by
Mobile phase A and Mobile phase B composition, volumn concentration of the mobile phase A in eluent is by 100% linear decline to 0%；The
45min eluent is Mobile phase B.
The HPLC spectrograms of amino acid standard liquid are as shown in Figure 2.In Fig. 2, Nle peak area is 1301274, Phe peak
Area is 3700021, and retention time is 33.65min.
The HPLC spectrograms of the dilution of Escherichia coli MD1078 fermented liquid supernatants are as shown in Figure 3.In Fig. 3, Nle peak area
It is 7746327 for 1301520, Phe peak area, retention time is 33.6min.
The HPLC spectrograms of the dilution of the fermented liquid supernatant of Escherichia coli 10245 are as shown in Figure 4.In Fig. 4, Nle peak area
It is 1873312 for 1302058, Phe peak area, retention time is 33.6min.
It is 86.5g/L, Escherichia coli to calculate the L-phenylalanine concentration obtained in Escherichia coli MD1078 fermented liquid supernatants
L-phenylalanine concentration in 10245 fermented liquid supernatants is 20.8g/L, and Escherichia coli MD1078 produces L- than Escherichia coli 10245
Phenylalanine improves about 315%.
The average production acid of the bacterial strain of current domestic production L-phenylalanine（L-phenylalanine）Typically in 62g/L, large intestine bar
Bacterium MD1078 has significant production L-phenylalanine ability, there is very high production application value.
Claims (10)
1. one plant of recombination bacillus coli MD1078 (Escherichia coli MD1078), its deposit number is CCTCC NO:M
2013509。
2. deposit number is CCTCC NO:Application of M 2013509 Escherichia coli in production L-phenylalanine.
3. a kind of method for producing L-phenylalanine, comprises the following steps：It is CCTCC NO by deposit number:M's 2013509
Escherichia coli fermentation culture, obtains L-phenylalanine.
4. method according to claim 3, it is characterised in that：The fermentation medium used in the fermented and cultured is every
100ml composition is as follows：
Glucose 2g, ammonium sulfate 2g, sodium citrate 0.05g, magnesium sulfate 0.08g, potassium dihydrogen phosphate 0.1g, dipotassium hydrogen phosphate
0.1g, dusty yeast 0.1g, sodium glutamate 0.05g, MnCl20.2mg, niacin 1mg, CoCl20.01mg, ZnSO40.1mg,
CaCl20.5mg, VB11mg, MnCl20.2mg, tyrosine 3mg, surplus is water.
5. the method according to claim 3 or 4, it is characterised in that：The condition of the fermentation is 36 DEG C, dissolved oxygen 35%-
50%, start to add glucose when the glucose content in fermentation system is less than 15g/L, be maintained at the content of glucose
Between 10g/L-15g/L.
6. method according to claim 5, it is characterised in that：The time of the fermentation is 48 hours.
7. the method according to claim 3 or 4, it is characterised in that：The deposit number is CCTCC NO:M 2013509
Escherichia coli be by seed liquor access fermentation medium.
8. method according to claim 7, it is characterised in that：The composition for preparing the every 100ml of culture medium of the seed liquor
It is as follows：Glucose 3g, ammonium sulfate 1g, magnesium sulfate 0.08g, potassium dihydrogen phosphate 0.1g, dipotassium hydrogen phosphate 0.1g, dusty yeast 0.2g,
Sodium glutamate 0.08g, MnCl20.25mg, CoCl20.005mg, ZnSO40.15mg, CaCl20.5mg, VB11mg, MnCl2
0.2mg, tyrosine 3mg, surplus is water.
9. method according to claim 7, it is characterised in that：Condition of culture when prepared by the seed liquor is 36 DEG C, molten
Oxygen is 40%-60%, and the content of glucose is 30g/L.
10. method according to claim 9, it is characterised in that：The incubation time of the seed liquor is 12 hours.
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| CN101717769A (en) * | 2009-12-08 | 2010-06-02 | 福建省麦丹生物集团有限公司 | Method for improving acid production rate of L-phenylalanine gene engineering bacteria |
| CN102604882A (en) * | 2012-03-31 | 2012-07-25 | 福建省麦丹生物集团有限公司 | Engineering bacterium for producing L-phenylalanine and application thereof |
| CN103146773A (en) * | 2013-03-08 | 2013-06-12 | 江南大学 | Method for enhancing L-phenylalanine exocytosis of escherichia coli |
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| CN101717769A (en) * | 2009-12-08 | 2010-06-02 | 福建省麦丹生物集团有限公司 | Method for improving acid production rate of L-phenylalanine gene engineering bacteria |
| CN102604882A (en) * | 2012-03-31 | 2012-07-25 | 福建省麦丹生物集团有限公司 | Engineering bacterium for producing L-phenylalanine and application thereof |
| CN103146773A (en) * | 2013-03-08 | 2013-06-12 | 江南大学 | Method for enhancing L-phenylalanine exocytosis of escherichia coli |
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