CN104745520B - A kind of strain excellent of high yield L phenylalanines and its application - Google Patents

A kind of strain excellent of high yield L phenylalanines and its application Download PDF

Info

Publication number
CN104745520B
CN104745520B CN201310752813.1A CN201310752813A CN104745520B CN 104745520 B CN104745520 B CN 104745520B CN 201310752813 A CN201310752813 A CN 201310752813A CN 104745520 B CN104745520 B CN 104745520B
Authority
CN
China
Prior art keywords
phenylalanine
fermentation
escherichia coli
phenylalanines
phea
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201310752813.1A
Other languages
Chinese (zh)
Other versions
CN104745520A (en
Inventor
吴伟斌
黄建忠
施巧琴
翁雪清
黄钦耿
赵燕玉
陈炳生
吴松刚
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fujian Maidan Biology Group Co., Ltd.
Fujian Normal University
Original Assignee
Fujian Maidan Biology Group Co ltd
Fujian Normal University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fujian Maidan Biology Group Co ltd, Fujian Normal University filed Critical Fujian Maidan Biology Group Co ltd
Priority to CN201310752813.1A priority Critical patent/CN104745520B/en
Publication of CN104745520A publication Critical patent/CN104745520A/en
Application granted granted Critical
Publication of CN104745520B publication Critical patent/CN104745520B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
    • C12P13/222—Phenylalanine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/01054—3-Deoxy-7-phosphoheptulonate synthase (2.5.1.54)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/01—Hydro-lyases (4.2.1)
    • C12Y402/01051—Prephenate dehydratase (4.2.1.51)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y504/00—Intramolecular transferases (5.4)
    • C12Y504/99—Intramolecular transferases (5.4) transferring other groups (5.4.99)
    • C12Y504/99005—Chorismate mutase (5.4.99.5)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses a kind of strain excellent of high yield L phenylalanines and its application.The entitled Escherichia coli MD8357 of the bacterial strain, its deposit number are CCTCC NO：M2013510.In 200L automatic fermenters, 37 DEG C, dissolved oxygen 30% 60%, the glucose content in fermentation system are maintained at 8g/L 10g/L, and fermentation time is that the L phenylalanine yield of the bacterial strain reaches 89.5g/L under conditions of 48 hours.The ability of Escherichia coli MD8357 productions L phenylalanines disclosed by the invention is high, beneficial to large scale fermentation, is had broad application prospects in the actual production of L phenylalanines.

Description

A kind of strain excellent of high yield L-phenylalanine and its application
Technical field
The present invention relates to a kind of strain excellent of high yield 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 amino acid necessary greatly of the eight of synthesis, is widely used in the fields such as food, feed, medicine and cosmetics.L-phenylalanine is made To synthesize Aspartame（Aspartame）One of two kinds of raw materials, preparing the dipeptide sweetener A Siba of low in calories, high sugariness Application in sweet tea is increasingly extensive, and the market demand of L-phenylalanine increases sharply.Other L-phenylalanine is also antineoplastic The required raw material of thing and amino acid transfusion preparation, with its continually developing in field of medicaments, demand is also continuously increased.Cause This, the improvement of research, L-phenylalanine producing strains to L-phenylalanine biosynthesis pathway and grinds to its industrialized production Study carefully and be increasingly 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 substituted 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 Produce the main stream approach of L-phenylalanine both at home and abroad at present, there is larger competitive advantage.But due to L-phenylalanine etc. eventually 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.
In the building process of L-phenylalanine genetic engineering bacterium, if recombinant plasmid is unstable, i.e., in recombinant bacterium culture Undergo mutation or lack in journey, recombinant bacterium will be made to lose original phenotypic characteristic.If the plasmid copy number that cell contains It is too high, or with the gene to host cell toxic side effect, and culture in a manner of by High Density Cultivation or continuously cultivating During engineering bacteria, the Loss Rate of plasmid can greatly increase.
In-vitro directed transformation is a kind of new molecular modification strategy, by manual simulation's natural evolution mechanism come realize " Evolution in test tube ".Its specific method is：Modifying gene simulates the random mutation of natural evolution and homologous heavy in vitro first Group, and under the selection pressure artificially created, filter out required property enters chemoattractant molecule.It is not required to understand in advance the sky of enzyme molecule Between structure and catalyst mechanism, in brief, orthogenesis=random mutation+positive restructuring+selection (or screening).Thus can be Very long natural evolution process is completed in short period, it might even be possible to create optimization within the time of a few weeks or months Enzyme, and in natural evolutionary process, obtaining this result needs several ten million years as long as.
The content of the invention
It is an object of the invention to provide a kind of strain excellent of high yield L-phenylalanine and its application.
One plant of recombination bacillus coli MD8357 provided by the invention, its deposit number are CCTCC NO：M2013510.
Deposit number is CCTCC NO：Application of the M2013510 Escherichia coli in L-phenylalanine is produced falls within this The protection domain of invention.
A kind of method for producing L-phenylalanine falls within protection scope of the present invention, comprises the following steps：Preservation is compiled Number it is CCTCC NO：M2013510 Escherichia coli carry out fermented and cultured, obtain L-phenylalanine.
In the above method, the every 100ml of fermentation medium used in the fermented and cultured 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 are water.
In any of the above-described described method, the condition of the fermentation is 37 DEG C, dissolved oxygen 30%-60%, in glucose during fermentation Content starts to add glucose when being less than 10g/L, the content of glucose is maintained between 8g/L-10g/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 are water.
In any of the above-described described method, condition of culture when prepared by the seed liquor is 36 DEG C, dissolved oxygen 40%-60%, The content of glucose is set to remain 30g/L during fermentation.
In any of the above-described described method, the incubation time of the seed liquor is 12 hours.
It is it is demonstrated experimentally that reachable using the bacterial strain production fermentation L-phenylalanine of the present invention, the yield of L-phenylalanine 89.5g/L, it was demonstrated that the ability of bacterial strain production L-phenylalanine of the invention is high, beneficial to large scale fermentation, in the reality of L-phenylalanine Had broad application prospects in the production of border.
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 MD8357 fermented liquid supernatants.
Fig. 4 is the HPLC spectrograms of the dilution of control strain FS001 fermented liquid supernatants.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
Ethyl methane sulfonate (ethyl methane sulfonate, EMS) is purchased from magnificent one bio tech ltd in Shanghai, Production number HQ0160.
PMD-18T is purchased from precious bioengineering（Dalian）Co., Ltd, catalog number D101A.
PBV220 is purchased from Beijing DingGuo ChangSheng Biology Technology Co., Ltd.
Escherichia coli（Escherichia coli）Purchased from Chinese industrial Microbiological Culture Collection administrative center （www.china-cicc.org）, numbering 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, production code member 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 to be settled to 100ml, sterilize.
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 to be settled to 100ml, sterilize.
Amino acid standard liquid is purchased from Tianjin Bonaaijieer Technology Co., Ltd.
Embodiment 1, produce L-phenylalanine bacterial strain preparation
First, recombinant plasmid pBV220-aroG-pheA structure
It is primer with primer aroG-f and aroG-r using the genomic DNA of Escherichia coli 10245 as template, enters performing PCR expansion Increase, obtain aroG genes.
It is primer with primer pheA-f and pheA-r using the genomic DNA of Escherichia coli 10245 as template, enters performing PCR expansion Increase, obtain pheA genes.
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）
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, is named as pMD-18T-aroG.
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, is named as pMD-18T-pheA.
PMD-18T-aroG is subjected to double digestion with Kpn I and BamH I, reclaims carrier large fragment；PMD-18T-pheA is used Kpn I and BamH I carries out double digestion, reclaims pheA genetic fragments；Carrier large fragment is connected with pheA genetic fragments, obtains weight Group plasmid, is named as 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, reclaims aroG-pheA genetic fragments；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 connects, and obtains recombinant plasmid, is named as pBV220-aroG-pheA, as shown in figure 1, sequence verification is correct.
2nd, recombinant plasmid pBV220-aroG-pheA EMS mutation
Ethyl methane sulfonate (ethyl methane sulfonate, EMS) is conventional alkylation chemistry mutagens.With step Rapid two obtained expression plasmid pBV220-aroG-pheA are mutated as mutation object with EMS methods to DNA.
Concretely comprise the following steps：Five 1.5ml EP pipes are taken, 1.5 μ g purifying expression plasmids is separately added into, it is high to be dissolved in 400 μ l respectively Pure water, after adding 4 μ 1EMS mixings, in 36 DEG C of warm bath 0min, 30min, 60min, 75min, 90min, sudden change conditions are selected Select, it is determined that optimal mutation time is 60min.
3rd, the mutant bacterium storehouse containing pBV220-aroG-pheA and control bacterium are built
EMS is mutated to processing 60min expression vector plasmid electricity conversion Escherichia coli 10245, obtained containing pBV220- AroG-pheA mutant bacterium storehouse.
By untreated carrier pBV220-aroG-pheA electricity conversion Escherichia coli 10245, control strain is obtained, is ordered Entitled FS001.
4th, screening obtains the L-phenylalanine base of high and stable yields from the mutant bacterium storehouse containing pBV220-aroG-pheA Because of engineered strain
（One）Preliminary screening
Bacterial strain in bacterium storehouse is recovered, is coupled with according to 4% inoculum concentration containing 3mg/ml, 4mg/ml, 5mg/ml, 6mg/ In the LB fluid nutrient mediums of the g/mL concentration of μ containing Amp100 of ml, 7mg/ml, 8mg/ml P-fluoropnenylalanine, 37 DEG C, 150rpm mistakes Night cultivates.
The bacterial strain in bacterium storehouse can be 3mg/ml, 4mg/ml, 5mg/ml, 6mg/ml, 7mg/ml in P-fluoropnenylalanine concentration Grown in the LB fluid nutrient mediums of the g/mL concentration of μ containing Amp100, but it is dense in the g/mL of μ containing Amp100 of 8mg/ml P-fluoropnenylalanine Growth is had no in degree LB fluid nutrient mediums.And contain the control strain of the pBV220-aroG-pheA plasmids without EMS mutation FS001, just have no and grown in the g/mL concentration LB fluid nutrient mediums of μ containing Amp100 that P-fluoropnenylalanine concentration is 4mg/ml.
The mutation library bacterium that will be grown in the 7mg/ml P-fluoropnenylalanine g/mL concentration LB fluid nutrient mediums of μ containing Amp100 Strain, carry out plate streaking culture and separation（Plating medium is to the ammonia of fluorobenzene third, the g/mL concentration of μ containing Amp100 LB containing 7mg/ml Solid medium）, obtain single bacterium colony.
（Two）Further screening
Picking step（One）Monoclonal to every 150 μ L of hole g/mL concentration of μ containing Amp100 fermentation medium 96 orifice plates It is interior, 37 DEG C, 150rpm culture 48h, 3000rpm centrifugations, supernatant is taken, it is 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 are defined between 0.2-0.7）, 4000r/min centrifugations 15 minutes.
3rd, each supernatant is taken to dilute 1000 times with 0.1mol/L hydrochloric acid solutions again, using 0.1mol/L hydrochloric acid as blank, measure Survey absorbance A206, A208, A222。
4th, the L-phenylalanine concentration in Sample supernatants is calculated according to formula 1（g/100ml）.
（Formula 1）
△ 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
From the bacterial strain of the storehouse of plant mutant more than 10,000, screening obtains 1 plant height production L-phenylalanine bacterial strain, and orifice plate yield is up to 0.956g/100mL bacterial strain, Escherichia coli MD8357 is denoted as, the orifice plate L-phenylalanine yield of other bacterial strains is in 0.28g/ Between 100mL to 0.89g/100mL.It is 0.32g/100mL that bacterium FS001, which is compareed, in the L-phenylalanine yield of orifice plate.
Escherichia coli MD8357（Escherichia coli MD8357）Chinese Typical Representative is preserved on October 25th, 2013 Culture collection（Abbreviation CCTCC, address：Chinese Wuhan Wuhan Universitys, postcode 430072）, deposit number CCTCC NO：M2013510.
The plasmid stability detection of embodiment 2, bacterial strain MD8357 and control strain FS001
By Escherichia coli MD8357 and control strain FS001 single bacterium colony access 2ml μ containing Amp100 g/mL LB culture mediums In, 35 DEG C of overnight incubations, by LB culture mediums of the seed liquor 20ul accesses without Amp antibiotic, 35 DEG C of cultures, make bacterial strain in nothing 48h is continuously grown in Amp culture medium, more than generation, last time, bacterium is growing into OD for breeding 50600When=0.3,37 are transferred to DEG C induction, sampling be applied on the LB flat boards without Amp, next day, random 100 points of picking colony are planted in the g/mL's of μ containing Amp100 On LB flat boards, bacterial strain MD8357 grown on the LB flat boards containing Amp bacterium colony percentage be 99%, control strain FS001 containing The percentage that bacterium colony is grown on Amp LB flat boards is 75%, it was demonstrated that Escherichia coli MD8357 has very high plasmid stability.
The 200L fermentation tank acid producing abilities of embodiment 3, Escherichia coli MD8357 and control strain FS001
First, seed culture（Using 50L automatic fermenters, seed culture medium liquid amount is 30L）：By strain Escherichia coli MD8357 and control strain FS001 are inoculated in seed culture medium respectively, and 50L 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 OD610nm=35）.
2nd, fermented and cultured（Using 200L automatic fermenters, fermentation liquid culture medium liquid amount is 120L）：Sent out using 200L Fermentation tank is fermented.Seed liquor is inoculated in fermentation medium by 25% volume, fermentation tank control parameter is 37 DEG C, dissolved oxygen （DO）30%-60%, when the glucose content in fermentation system is less than 10g/L, add 700g/L glucose automatically by fermentation tank The aqueous solution so that the glucose content of fermentation system is maintained between 8g/L-10g/L, and fermentation time is 48 hours.
After Escherichia coli MD8357 and control strain FS001 48 hours zymotic fluid 15mL4500rpm centrifugations, its hair is taken Ferment supernatant carries out liquid phase detection after being diluted with water to 200 times of volumes.
3rd, liquid phase detects the content of L-phenylalanine in fermented liquid supernatant
L-phenylalanine（phe）The assay method of concentration often uses assay method, i.e. high performance liquid chromatography using amino acid （HPLC）Derivatization Method（Rapid,accμrate,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 of L-phenylalanine in f1=sample solution Area/interior target peak area；The peak area of L-phenylalanine/interior target peak area in f2=amino acid standard liquid；C=amino acid L-phenylalanine in 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, mix, 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, add 10mL0.1moL/L aqueous hydrochloric acid solutions, dissolve；
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, carry out as follows：
（1）Amino acid standard liquid and each 200 μ L of analyte sample fluid are measured respectively, 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（PTC- phenylalanines, the phenylalanine after deriving）, filtered with 0.45 μm of pin type filter；
（6）The μ L of filtrate 200 are taken, adds 800 μ L water to dilute, shakes 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 3700438, retention time 33.65min.
The HPLC spectrograms of the dilution of Escherichia coli MD8357 fermented liquid supernatant are as shown in Figure 3.In Fig. 3, Nle peak face The peak area that product is 1301520, phe is 8011254, retention time 33.6min.
The HPLC spectrograms of the dilution of control strain FS001 fermented liquid supernatant are as shown in Figure 4.In Fig. 4, Nle peak face The peak area that product is 1302058, phe is 3745312, retention time 33.6min.
The L-phenylalanine concentration that Escherichia coli MD8357 fermented liquid supernatant is calculated is 89.5g/L, control strain The L-phenylalanine concentration of FS001 fermented liquid supernatant is 41.8g/L, and MD8357 is improved about than FS001 production L-phenylalanines 114%。
Average production acid of the bacterial strain of domestic production L-phenylalanine in 200L fermentation tanks at present（L-phenylalanine）Typically exist 65g/L, Escherichia coli MD8357 have significant production L-phenylalanine ability, there is very high production application value.

Claims (2)

1. one plant of recombination bacillus coli MD8357, its deposit number is CCTCC NO：M 2013510.
2. deposit number is CCTCC NO：Application of M 2013510 Escherichia coli in L-phenylalanine is produced.
CN201310752813.1A 2013-12-31 2013-12-31 A kind of strain excellent of high yield L phenylalanines and its application Active CN104745520B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201310752813.1A CN104745520B (en) 2013-12-31 2013-12-31 A kind of strain excellent of high yield L phenylalanines and its application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201310752813.1A CN104745520B (en) 2013-12-31 2013-12-31 A kind of strain excellent of high yield L phenylalanines and its application

Publications (2)

Publication Number Publication Date
CN104745520A CN104745520A (en) 2015-07-01
CN104745520B true CN104745520B (en) 2018-02-23

Family

ID=53585781

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201310752813.1A Active CN104745520B (en) 2013-12-31 2013-12-31 A kind of strain excellent of high yield L phenylalanines and its application

Country Status (1)

Country Link
CN (1) CN104745520B (en)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108841733B (en) * 2018-07-26 2021-09-28 福州工微生物科技有限公司 Strain and method for producing griseofulvin serving as major component of tranexamycin
CN108841889B (en) * 2018-07-26 2021-09-28 福州工微生物科技有限公司 Method for producing griseofulvin serving as major component of tranexamycin by microbial fermentation
CN108823110B (en) * 2018-07-26 2021-07-16 福州工微生物科技有限公司 Strain for producing griseofulvin and application thereof
CN112251476B (en) * 2020-09-25 2022-11-15 天津科技大学 Production method of L-phenylalanine
CN113801901B (en) * 2021-07-30 2024-05-24 新泰市佳禾生物科技有限公司 Method for producing L-phenylalanine by fermentation

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101698853A (en) * 2009-03-31 2010-04-28 福建省麦丹生物集团有限公司 In-vitro directed coevolution method for modifying L-phenylalanine gene engineering strains
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

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101698853A (en) * 2009-03-31 2010-04-28 福建省麦丹生物集团有限公司 In-vitro directed coevolution method for modifying L-phenylalanine gene engineering strains
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

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
L-苯丙氨酸基因工程菌的分子育种研究进展;吴伟斌等;《发酵通讯科技》;20120131;第41卷(第1期);27-29 *

Also Published As

Publication number Publication date
CN104745520A (en) 2015-07-01

Similar Documents

Publication Publication Date Title
CN104745520B (en) A kind of strain excellent of high yield L phenylalanines and its application
CN107267578A (en) The method that microbial fermentation produces N acetyl D Glucosamines and/or D glucosamine salts
CN109402158A (en) A kind of recombinant expression plasmid carrier, metabolic engineering bacteria and production method producing fucosyllactose
CN103409485A (en) Method for improving adenosine fermentation output through feeding organic nitrogen source
CN102994439A (en) Escherichia coli recombinant strain producing shikimic acid, and construction method and application thereof
CN113755422B (en) Recombinant amycolatopsis capable of highly producing vanillin, construction method and application thereof
CN110591989A (en) High-yield L-tryptophan engineering strain and application thereof
CN105420154A (en) Double knockout recombinant rhodococcus as well as construction method and application thereof
CN102010847B (en) Antiphagin L-phenylalanine producing strain as well as breeding method and application thereof
CN107916283A (en) A kind of production technology of niacinamide
CN105695351B (en) S.actuosus LB-16 and the method for preparing Nosiheptide using it
CN110257313B (en) Engineering bacterium of bacillus amyloliquefaciens for producing spermidine
CN103146773B (en) Method for enhancing L-phenylalanine exocytosis of escherichia coli
CN109234337B (en) Biosynthesis method of quercetin glycoside
CN110257448A (en) A method of using thallus whole-cell catalytic conversion of Arginine be gamatine
CN113789292B (en) Gene-deficient amycolatopsis capable of producing vanillin at high yield, and construction method and application thereof
CN113817757B (en) Recombinant yeast engineering strain for producing cherry glycoside and application thereof
CN101948764B (en) Pseudomonas syringae pv.mori bacterial strain for producing coronatine and method for producing coronatine by fermentation thereof
CN111041020B (en) Isocitrate lyase mutant, mutant gene and application thereof in preparation of vitamin B12In (1)
CN106010999A (en) Gene engineering strain, culturing method and application of gene engineering strain
CN104745500B (en) One plant production L phenylalanines bacterial strain and its application
CN113881615A (en) Xenorhabdus nematophila with high yield of Xcn1 and application thereof
CN112941002A (en) Recombinant Escherichia coli strain for producing dopamine and construction method and application thereof
KR20140007425A (en) Method for preparing cyclic lipopeptide compound
CN109097374A (en) A kind of preparation method, bacterial strain and its application of pseudomonas aeruginosa engineered strain

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
CB03 Change of inventor or designer information

Inventor after: Wu Weibin

Inventor after: Huang Jianzhong

Inventor after: Shi Qiaoqin

Inventor after: Weng Xueqing

Inventor after: Huang Qingeng

Inventor after: Zhao Yanyu

Inventor after: Chen Bingsheng

Inventor after: Wu Songgang

Inventor before: Shi Qiaoqin

Inventor before: Wu Weibin

Inventor before: Huang Qingeng

Inventor before: Weng Xueqing

Inventor before: Zhao Yanyu

Inventor before: Huang Xiangfeng

Inventor before: Wu Songgang

CB03 Change of inventor or designer information
TA01 Transfer of patent application right

Effective date of registration: 20180118

Address after: 350007 Fuzhou Road, Cangshan District, Fujian, No. three on the road 8

Applicant after: Fujian Normal University

Applicant after: Fujian Maidan Biology Group Co., Ltd.

Address before: 365500 Jinsha hi tech park, Fujian, Sanming City Province

Applicant before: Fujian Maidan Biology Group Co., Ltd.

TA01 Transfer of patent application right
GR01 Patent grant
GR01 Patent grant