CN104745520A - Excellent strain capable of high-yielding L-phenylalanine and application of excellent strain - Google Patents
Excellent strain capable of high-yielding L-phenylalanine and application of excellent strain Download PDFInfo
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Abstract
The invention discloses an excellent strain capable of high-yielding L-phenylalanine and application of the excellent strain. The strain is named as escherichia coli MD8357, and the preservation serial number is CCTCC NO:M2013510. Under the conditions that in a full-automatic fermentation tank of 200L, the temperature is 37 DEG C dissolved oxygen content is 30-60%, the content of glucose in the fermentation system is kept being 8g-10g/L, and the fermentation time is 48 hours, the yield of L-phenylalanine of the strain is 89.5g/L. The escherichia coli MD8357 disclosed by the invention is high in yield of L-phenylalanine, is beneficial for large-scale fermentation, and has wide application prospect in practical production of L-phenylalanine.
Description
Technical field
The present invention relates to a kind of strain excellent and application thereof of high yield L-Phe.
Background technology
L-Phe (L-phenylalanine) is white crystalline powder, be human body and animal can not synthesize voluntarily in vivo eight greatly must one of amino acid, be widely used in the fields such as food, feed, medicine and makeup.L-Phe is as one of two kinds of raw materials synthesizing aspartame (Aspartame), and the application in the dipeptide sweetener aspartame of low in calories, the high sugariness of preparation is increasingly extensive, and the market demand of L-Phe increases sharply.L-Phe is also the required raw material of antitumor drug and amino acid transfusion preparation in addition, and along with its continually developing at field of medicaments, demand also constantly increases.Therefore, to the research of L-Phe biosynthetic pathway, the improvement of L-Phe producing strains be more and more subject to people's attention the research of its suitability for industrialized production.
The production method of domestic and international L-Phe mainly contains hydrolysis extraction process, chemical synthesis, enzyme process and fermentation method etc.Because in native protein, L-Phe content is lower, hydrolysis extraction process seldom uses; The complex process of chemical synthesis, cost is higher, is substantially replaced by enzyme process and fermentation method abroad.But the higher and limited source of the price due to substrate and enzyme, enzyme process application is also restricted; Because microorganism direct fermentation can utilize raw material cheap and easy to get, can carry out at normal temperatures and pressures again, be the main stream approach of producing L-Phe at present both at home and abroad, there is larger competitive edge.But because the end products such as L-Phe have strong feedback inhibition to the key enzyme on the die aromatischen Aminosaeuren metabolic pathway of synthesizing in wild strain body, make its cell can not accumulate phenylalanine in a large number, therefore to carry out real attenuation production, just needing badly to obtain and producing the high bacterial strain of L-Phe ability.
In the building process of L-Phe genetic engineering bacterium, if recombinant plasmid is unstable, namely undergos mutation in recombinant bacterium culturing process or lack, recombinant bacterium will be made to lose original phenotypic characteristic.If the plasmid copy number that cell contains is too high, or with the gene to host cell toxic side effect, and during mode culturing engineering bacterium with high-density culture or cultured continuously, the Loss Rate of plasmid can increase greatly.
In-vitro directed transformation is a kind of new molecular modification strategy, is realized " evolution in test tube " by manual simulation's natural evolution mechanism.Its concrete grammar is: modifying gene carrys out random mutation and the homologous recombination of simulating nature evolution first in vitro, and under the artificial selective pressure created, filters out the evolution molecule of required character.It does not need space structure and the catalyst mechanism of understanding enzyme molecule in advance, in brief, and orthogenesis=random mutation+forward restructuring+select (or screening).So just can complete very long natural evolution process within a short period of time, even can create the enzyme of optimization within the time of a few weeks or months, and in natural evolutionary process, obtaining this result needs several ten million years.
Summary of the invention
The object of this invention is to provide a kind of strain excellent and application thereof of high yield L-Phe.
A strain recombination bacillus coli MD8357 provided by the invention, its deposit number is CCTCC NO:M2013510.
Deposit number is that the application of intestinal bacteria in production L-Phe of CCTCC NO:M2013510 also belongs to protection scope of the present invention.
The method of producing L-Phe also belongs to a protection scope of the present invention, comprises the steps: to be that the intestinal bacteria of CCTCC NO:M2013510 carry out fermentation culture by deposit number, obtains L-Phe.
In aforesaid method, the every 100ml's of the fermention medium used in described fermentation culture is composed as follows:
Glucose 2g, ammonium sulfate 2g, Trisodium Citrate 0.05g, magnesium sulfate 0.08g, potassium primary phosphate 0.1g, dipotassium hydrogen phosphate 0.1g, yeast powder 0.1g, Sodium Glutamate 0.05g, MnCl
20.2mg, nicotinic acid 1mg, CoCl
20.01mg, ZnSO
40.1mg, CaCl
20.5mg, V
b11mg, MnCl
20.2mg, tyrosine 3mg, surplus is water.
In above-mentioned arbitrary described method, the condition of described fermentation is 37 DEG C, dissolved oxygen 30%-60%, adds glucose during fermentation at glucose content lower than starting during 10g/L, and the content of glucose is remained between 8g/L-10g/L.
In above-mentioned arbitrary described method, the time of described fermentation is 48 hours.
In above-mentioned arbitrary described method, described bacterial strain is by seed liquor access fermention medium.
In above-mentioned arbitrary described method, described seed liquor prepare the composed as follows of the every 100ml of substratum: glucose 3g, ammonium sulfate 1g, magnesium sulfate 0.08g, potassium primary phosphate 0.1g, dipotassium hydrogen phosphate 0.1g, yeast powder 0.2g, Sodium Glutamate 0.08g, MnCl
20.25mg, CoCl
20.005mg, ZnSO
40.15mg, CaCl
20.5mg, V
b11mg, MnCl
20.2mg, tyrosine 3mg, surplus is water.
In above-mentioned arbitrary described method, culture condition time prepared by described seed liquor is 36 DEG C, dissolved oxygen is 40%-60%, makes the content of glucose remain 30g/L during fermentation.
In above-mentioned arbitrary described method, the incubation time of described seed liquor is 12 hours.
Experiment proves, utilize bacterial strain of the present invention to produce fermentation L-Phe, the output of L-Phe can reach 89.5g/L, proves that the ability of bacterial strain of the present invention product L-Phe is high, be beneficial to large scale fermentation, have broad application prospects in the actual production of L-Phe.
Accompanying drawing explanation
Fig. 1 is pBV220-aroG-pheA plasmid.
Fig. 2 is the HPLC spectrogram of amino acid standardized solution.
Fig. 3 is the HPLC spectrogram of the diluent of intestinal bacteria MD8357 fermented liquid supernatant.
Fig. 4 is the HPLC spectrogram of the diluent of control strain FS001 fermented liquid supernatant.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Ethyl methane sulfonate (ethyl methane sulfonate, EMS) purchased from Shanghai China one bio tech ltd, production number HQ0160.
PMD-18T is purchased from precious biotechnology (Dalian) company limited, and catalog number is D101A.
PBV220 is purchased from Beijing DingGuo ChangSheng Biology Technology Co., Ltd.
Intestinal bacteria (Escherichia coli), purchased from Chinese industrial Microbiological Culture Collection administrative center (www.china-cicc.org), are numbered 10245, are called for short intestinal bacteria 10245.
Fermention medium: 50g/L glucose, 10g/L yeast extract paste, 3g/L Na
2hPO
4, 1g/L KH
2pO
4, 12g/L NH
4cl, 1g/L MgSO
4, 0.3g/L CaCl
2, 6g/L Trisodium Citrate, 0.6g/L Sodium Glutamate, 2g/L peptone, 20mg/LV
b1, surplus is water.
L-Phe is full of Ze Naxin Chemical Engineering Technology research institute purchased from Beijing, and production code member is GF2212.
Every 100ml fermentor tank seed culture medium is prepared as follows:
Glucose 3g, ammonium sulfate 1g, magnesium sulfate 0.08g, potassium primary phosphate 0.1g, dipotassium hydrogen phosphate 0.1g, yeast powder 0.2g, Sodium Glutamate 0.08g, MnCl
20.25mg, CoCl
20.005mg, ZnSO
40.15mg, CaCl
20.5mg, V
b11mg, MnCl
20.2mg, tyrosine 3mg, adds water and is settled to 100ml, sterilizing.
Every 100ml ferment tank substratum is prepared as follows:
Glucose 2g, ammonium sulfate 2g, Trisodium Citrate 0.05g, magnesium sulfate 0.08g, potassium primary phosphate 0.1g, dipotassium hydrogen phosphate 0.1g, yeast powder 0.1g, Sodium Glutamate 0.05g, MnCl
20.2mg, nicotinic acid 1mg, CoCl
20.01mg, ZnSO
40.1mg, CaCl
20.5mg, V
b11mg, MnCl
20.2mg, tyrosine 3mg, adds water and is settled to 100ml, sterilizing.
Amino acid standardized solution is purchased from Tianjin Bonaaijieer Technology Co., Ltd.
The preparation of the bacterial strain of embodiment 1, production L-Phe
One, the structure of recombinant plasmid pBV220-aroG-pheA
With the genomic dna of intestinal bacteria 10245 for template, be primer with primer aroG-f and aroG-r, carry out pcr amplification, obtain aroG gene.
With the genomic dna of intestinal bacteria 10245 for template, be primer with primer pheA-f and pheA-r, carry out pcr amplification, obtain pheA gene.
aroG-f:5'-CG
GAATTCATGAATTATCAGAACGACGAT-3'
aroG-r:5'-GG
GGTACCACCCGCGACGCGCTTTTACTG-3'
pheA-f:5'-GG
GGTACCATGACATCGGAAAACCCGTTAC-3'
pheA-r:5'-CG
GGATCCTCAGGTTGGATCAACAGGCAC-3'
(sequence shown in underscore is that enzyme cuts recognition site)
EcoR I and Kpn I double digestion aroG gene, obtain gene fragment; EcoR I and Kpn I double digestion pMD-18T, obtains carrier large fragment; Gene fragment is connected with carrier large fragment, obtains recombinant plasmid, by its called after pMD-18T-aroG.
Kpn I and BamH I double digestion pheA gene, obtain gene fragment; Kpn I and BamH I double digestion pMD-18T, obtains carrier large fragment; Gene fragment is connected with carrier large fragment, obtains recombinant plasmid, by its called after pMD-18T-pheA.
PMD-18T-aroG Kpn I and BamH I is carried out double digestion, reclaims carrier large fragment; PMD-18T-pheA Kpn I and BamH I is carried out double digestion, reclaims pheA gene fragment; Carrier large fragment is connected with pheA gene fragment, obtains recombinant plasmid, called after pMD-aroG-pheA.PMD-aroG-pheA is carried out sequence verification, and result is correct.
Plasmid pMD-aroG-pheA EcoR I and BamH I is carried out double digestion, reclaims aroG-pheA gene fragment; Plasmid pBV220 EcoR I and BamH I is carried out double digestion, reclaims carrier large fragment; Carrier large fragment be connected with aroG-pheA gene fragment, obtain recombinant plasmid, called after pBV220-aroG-pheA, as shown in Figure 1, sequence verification is correct.
Two, the EMS sudden change of recombinant plasmid pBV220-aroG-pheA
Ethyl methane sulfonate (ethyl methane sulfonate, EMS) is conventional alkylation chemistry mutagens.The expression plasmid pBV220-aroG-pheA obtained using step 2, as sudden change object, suddenlys change to plasmid DNA by EMS method.
Concrete steps are: get five 1.5ml EP and manage, add 1.5 μ g purifying expression plasmids respectively, be dissolved in 400 μ l high purity waters respectively, after adding 4 μ 1EMS mixings, in 36 DEG C of temperature bath 0min, 30min, 60min, 75min, 90min, select sudden change conditions, determines that best mutation time is 60min.
Three, the mutant bacterium storehouse containing pBV220-aroG-pheA and contrast bacterium is built
By the expression vector plasmid electricity transformation of E. coli 10245 of EMS sudden change process 60min, obtain the mutant bacterium storehouse containing pBV220-aroG-pheA.
By untreated carrier pBV220-aroG-pheA electricity transformation of E. coli 10245, obtain control strain, by its called after FS001.
Four, from the mutant bacterium storehouse containing pBV220-aroG-pheA, screening obtains the L-Phe engineering strain of high and stable yields
(1) preliminary screening
By in bacterium storehouse bacterial strain recovery, the inoculum size according to 4% receive respectively containing 3mg/ml, 4mg/ml, 5mg/ml, 6mg/ml, 7mg/ml, 8mg/ml P-fluoropnenylalanine containing Amp100 μ g/mL concentration LB liquid nutrient medium in, 37 DEG C, 150rpm incubated overnight.
The bacterial strain in bacterium storehouse can be grow in P-fluoropnenylalanine concentration containing in the LB liquid nutrient medium of Amp100 μ g/mL concentration of 3mg/ml, 4mg/ml, 5mg/ml, 6mg/ml, 7mg/ml, but has no growth in containing in Amp100 μ g/mL concentration LB liquid nutrient medium of 8mg/ml P-fluoropnenylalanine.And the control strain FS001 containing the pBV220-aroG-pheA plasmid without EMS sudden change, be that just having no containing Amp100 μ g/mL concentration LB liquid nutrient medium of 4mg/ml grown in P-fluoropnenylalanine concentration.
The mutation library bacterial strain grown in Amp100 μ g/mL concentration LB liquid nutrient medium will be contained at 7mg/ml P-fluoropnenylalanine, carry out plate loop method and be separated (plate culture medium is for containing 7mg/ml to fluorobenzene third ammonia, containing Amp100 μ g/mL concentration LB solid medium), obtain single bacterium colony.
(2) screen further
The mono-clonal of picking step () contains in 96 orifice plates of the fermention medium of Amp100 μ g/mL concentration to every hole 150 μ L, 37 DEG C, it is centrifugal that 150rpm cultivates 48h, 3000rpm, gets supernatant, to be measured.
Three wave bands survey the content that acid system detects L-Phe in fermented supernatant fluid:
1, prepare the 0.1g/100ml L-Phe aqueous solution as standard substance, then after diluting 1000 times with 0.1mol/L hydrochloric acid soln, with 0.1mol/L hydrochloric acid soln for blank, survey absorbance A
206, A
208, A
222.
2, fermentation broth sample supernatant distilled water diluting n doubly rear (in the Sample supernatants finally recorded, L-Phe should at about 0.1g/100ml, and each absorbancy is as the criterion between 0.2-0.7), centrifugal 15 minutes of 4000r/min.
3, get each supernatant liquor and dilute 1000 times with 0.1mol/L hydrochloric acid soln again, with 0.1mol/L hydrochloric acid for blank, measure and survey absorbance A
206, A
208, A
222.
4, according to the L-Phe concentration (g/100ml) in formula 1 calculation sample supernatant.
(formula 1)
△ sample=A
206 samples+ 0.2727A
208 samples-0.9682A
222 samples
△ mark=A
206 marks+ 0.2727A
208 marks-0.9682A
222 marks
Extension rate: 1000
Standard concentration: 0.1g/100mL
From the bacterial strain of the storehouse of plant mutant more than 10,000, screening acquisition 1 plant height produces L-Phe bacterial strain, orifice plate output, up to the bacterial strain of 0.956g/100mL, is denoted as intestinal bacteria MD8357, and the orifice plate L-Phe output of other bacterial strains is between 0.28g/100mL to 0.89g/100mL.Contrast bacterium FS001 is 0.32g/100mL in the L-Phe output of orifice plate.
Intestinal bacteria MD8357(Escherichia coli MD8357) be preserved in China typical culture collection center (abbreviation CCTCC on October 25th, 2013, address: China. Wuhan. Wuhan University, postcode 430072), deposit number is CCTCC NO:M2013510.
The plasmid stability of embodiment 2, bacterial strain MD8357 and control strain FS001 detects
By single bacterium colony access 2ml of intestinal bacteria MD8357 and control strain FS001 containing in the LB substratum of Amp100 μ g/mL, 35 DEG C of overnight incubation, by seed liquor 20ul access not containing in the antibiotic LB substratum of Amp, 35 DEG C of cultivations, bacterial strain is made to grow 48h continuously in without the substratum of Amp, breed 50 generations more than, for the last time, bacterium is growing into OD
600when=0.3, proceed to 37 DEG C of inductions, sampling is applied to not containing on the LB flat board of Amp, next day, random picking colony 100 points are planted on the LB flat board containing Amp100 μ g/mL, bacterial strain MD8357 is 99% at the percentage ratio of the LB grow on plates bacterium colony containing Amp, and control strain FS001 is 75% at the percentage ratio of the LB grow on plates bacterium colony containing Amp, proves that intestinal bacteria MD8357 has very high plasmid stability.
The 200L fermentor tank acid producing ability of embodiment 3, intestinal bacteria MD8357 and control strain FS001
One, seed culture (adopts 50L automatic fermenter, seed culture medium liquid amount is 30L): strain Escherichia coli MD8357 and control strain FS001 is inoculated in seed culture medium respectively, 50L seed fermentation tank controling parameters is 36 DEG C, dissolved oxygen (DO) is 40%-60%, in seed culture medium, glucose concn is 30g/L, incubation time is 12 hours, obtains seed liquor (seed liquor OD
610nm=35).
Two, fermentation culture (adopt 200L automatic fermenter, fermented liquid substratum liquid amount is 120L): use 200L fermentor tank to ferment.By seed liquor by 25% volume be inoculated in fermention medium, fermentor tank controling parameters is 37 DEG C, dissolved oxygen (DO) 30%-60%, when glucose content in fermentation system is lower than 10g/L, automatically the D/W of 700g/L is added by fermentor tank, make the glucose content of fermentation system remain between 8g/L-10g/L, fermentation time is 48 hours.
After centrifugal for the 48 hours fermentation liquid 15mL4500rpm of intestinal bacteria MD8357 and control strain FS001, get after its fermented supernatant fluid is diluted with water to 200 times of volumes and carry out Liquid Detection.
Three, the content of L-Phe in Liquid Detection fermented liquid supernatant
The measuring method of L-Phe (phe) concentration adopts amino acid to commonly use measuring method, i.e. high performance liquid chromatography (HPLC) Derivatization Method (Rapid, acc μ rate, senstitive, and reprodicobLe HPLCanaLysis of amino acida.Henderson, J.W, Ricker, R.D., BidLingmeyer, B.A., Woodward, C., AgiLent TechnoLogier, Μ SA, 2000).
L-Phe concentration (μ g/mL)=f1/f2 × C in sample solution; Peak area/interior target the peak area of L-Phe in f1=sample solution; Peak area/interior target the peak area of L-Phe in f2=amino acid standardized solution; L-Phe (μ g/mL) in C=amino acid standardized solution.
Chromatographic column: AgeLa VenusiL AA post, (4.6*250mm, 5 μm);
Column temperature: 40 DEG C;
Determined wavelength: 254nm;
Mobile phase A: get 15.2g sodium acetate, anhydrous, add water 1850mL, and adjust pH to 6.5 with Glacial acetic acid after dissolving, then add 140mL acetonitrile, mixing, with 0.45 μm of membrane filtration;
Mobile phase B: acetonitrile and water are prepared according to volume ratio 4:1;
Flow velocity: 1mL/min;
Nor-leucine inner mark solution: get nor-leucine (Nle) 10mg, add 10mL0.1moL/L aqueous hydrochloric acid, dissolves;
In amino acid standardized solution, phenyl-alanine concentration is 2.5 μm of oL/mL;
Amino acid standardized solution and sample solution derivative, carries out as follows:
(1) measure amino acid standardized solution and each 200 μ L of analyte sample fluid respectively, be placed in 1.5mL centrifuge tube respectively;
(2) nor-leucine inner mark solution 20 μ L is added in each centrifuge tube;
(3) add 100 μ L triethylamine acetonitrile solutions in each centrifuge tube and (in 1.4mL triethylamine, add 8.6mL acetonitrile, mixing) and 100 μ L thiocarbanil acetonitrile solutions (25 μ L thiocarbanils add 2mL acetonitrile, mixing), mixing, room temperature places 1 hour;
(4) add 400 μ L normal hexanes in each centrifuge tube, after jolting, place 10min;
(5) a layer solution (PTC-phenylalanine, the phenylalanine after derivative) is taken off, with 0.45 μm of pin type frit;
(6) get filtrate 200 μ L, add 800 μ L water dilutions, shake up, sample introduction 10 μ L.
The elutriant of gradient elution process: 0-2min is mobile phase A; The elutriant of 2-14min is made up of mobile phase A and Mobile phase B, and the volumn concentration of mobile phase A in elutriant linearly drops to 93% by 100%; The elutriant of 14-29min is made up of mobile phase A and Mobile phase B, and the volumn concentration of mobile phase A in elutriant linearly drops to 70% by 93%; The elutriant of 29-32min is made up of mobile phase A and Mobile phase B, and the volumn concentration of mobile phase A in elutriant linearly drops to 50% by 70%; The elutriant of 32-33min is made up of mobile phase A and Mobile phase B, and the volumn concentration of mobile phase A in elutriant linearly rises to 100% by 50%; The elutriant of 33-39min is mobile phase A; The elutriant of 39-45min is made up of mobile phase A and Mobile phase B, and the volumn concentration of mobile phase A in elutriant linearly drops to 0% by 100%; The elutriant of 45min is Mobile phase B.
The HPLC spectrogram of amino acid standardized solution as shown in Figure 2.In Fig. 2, the peak area of Nle is the peak area of 1301274, phe is 3700438, and retention time is 33.65min.
The HPLC spectrogram of the diluent of the fermented liquid supernatant of intestinal bacteria MD8357 as shown in Figure 3.In Fig. 3, the peak area of Nle is the peak area of 1301520, phe is 8011254, and retention time is 33.6min.
The HPLC spectrogram of the diluent of the fermented liquid supernatant of control strain FS001 as shown in Figure 4.In Fig. 4, the peak area of Nle is the peak area of 1302058, phe is 3745312, and retention time is 33.6min.
The L-Phe concentration calculating the fermented liquid supernatant of intestinal bacteria MD8357 is 89.5g/L, and the L-Phe concentration of the fermented liquid supernatant of control strain FS001 is that 41.8g/L, MD8357 produce L-Phe raising about 114% than FS001.
The bacterial strain of current domestic production L-Phe is general at 65g/L average product acid (L-Phe) of 200L fermentor tank, and intestinal bacteria MD8357 has significant product L-Phe ability, has very high production application to be worth.
Claims (10)
1. a strain recombination bacillus coli MD8357, its deposit number is CCTCC NO:M2013510.
2. deposit number is the application of intestinal bacteria in production L-Phe of CCTCC NO:M2013510.
3. produce a method for L-Phe, comprise the steps: to be that the intestinal bacteria of CCTCC NO:M2013510 carry out fermentation culture by deposit number, obtain L-Phe.
4. method according to claim 3, is characterized in that: the every 100ml's of the fermention medium used in described fermentation culture is composed as follows:
Glucose 2g, ammonium sulfate 2g, Trisodium Citrate 0.05g, magnesium sulfate 0.08g, potassium primary phosphate 0.1g, dipotassium hydrogen phosphate 0.1g, yeast powder 0.1g, Sodium Glutamate 0.05g, MnCl
20.2mg, nicotinic acid 1mg, CoCl
20.01mg, ZnSO
40.1mg, CaCl
20.5mg, V
b11mg, MnCl
20.2mg, tyrosine 3mg, surplus is water.
5. the method according to claim 3 or 4, is characterized in that: the condition of described fermentation is 37 DEG C, dissolved oxygen 30%-60%, adds glucose during fermentation at glucose content lower than starting during 10g/L, and the content of glucose is remained between 8g/L-10g/L.
6., according to the arbitrary described method of claim 3-5, it is characterized in that: the time of described fermentation is 48 hours.
7. according to the arbitrary described method of claim 3-6, it is characterized in that: described bacterial strain is by seed liquor access fermention medium.
8., according to the arbitrary described method of claim 3-7, it is characterized in that: described seed liquor prepare the composed as follows of the every 100ml of substratum: glucose 3g, ammonium sulfate 1g, magnesium sulfate 0.08g, potassium primary phosphate 0.1g, dipotassium hydrogen phosphate 0.1g, yeast powder 0.2g, Sodium Glutamate 0.08g, MnCl
20.25mg, CoCl
20.005mg, ZnSO
40.15mg, CaCl
20.5mg, V
b11mg, MnCl
20.2mg, tyrosine 3mg, surplus is water.
9., according to the arbitrary described method of claim 3-8, it is characterized in that: culture condition time prepared by described seed liquor is 36 DEG C, dissolved oxygen is 40%-60%, makes the content of glucose remain 30g/L during fermentation.
10., according to the arbitrary described method of claim 3-9, it is characterized in that: the incubation time of described seed liquor is 12 hours.
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| CN108823110A (en) * | 2018-07-26 | 2018-11-16 | 福州工微生物科技有限公司 | One plant of bacterial strain for producing griseofulvin and its application |
| CN108841733A (en) * | 2018-07-26 | 2018-11-20 | 福州工微生物科技有限公司 | The one plant of production main part of Songgangmeisu --- bacterial strain and method of griseofulvin |
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| 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 |
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| CN112251476A (en) * | 2020-09-25 | 2021-01-22 | 天津科技大学 | Production method of L-phenylalanine |
| CN112251476B (en) * | 2020-09-25 | 2022-11-15 | 天津科技大学 | Production method of L-phenylalanine |
| CN113801901A (en) * | 2021-07-30 | 2021-12-17 | 新泰市佳禾生物科技有限公司 | Method for producing L-phenylalanine by fermentation |
| 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 |
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