CN103146773B - Method for enhancing L-phenylalanine exocytosis of escherichia coli - Google Patents
Method for enhancing L-phenylalanine exocytosis of escherichia coli Download PDFInfo
- Publication number
- CN103146773B CN103146773B CN201310074890.6A CN201310074890A CN103146773B CN 103146773 B CN103146773 B CN 103146773B CN 201310074890 A CN201310074890 A CN 201310074890A CN 103146773 B CN103146773 B CN 103146773B
- Authority
- CN
- China
- Prior art keywords
- tyrb
- arog
- phea
- phenylalanine
- yddg
- 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
Links
Abstract
The invention discloses a method for enhancing the L-phenylalanine exocytosis of escherichia coli. The method comprises the steps of transforming host escherichia coli through recombinant plasmids capable of simultaneously overexpressing tyrosine aminotransferase TyrB, methyl viologen exo transport protein YddG, 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase AroG, chorismate mutase and prephenate dehydratase PheA, shikimate kinase AroK and shikimate dehydrogenase YdiB and producing L-phenylalanine through liquid fermentation, wherein artificial modification is carried out on TyrB, YddG, AroG, PheA, AroK and YdiB encoding genes from the escherichia coli resource so as to get release from the metabolic regulation and vectors are inserted, thus constructing the recombinant plasmids. The method disclosed by the invention has the advantages that the L-phenylalanine exocytosis level of the escherichia coli can be obviously enhanced, the L-phenylalanine yield is high, the concentration of harmful by-product acetic acid is low, the fermentation cost is low and the process is simple; and therefore, the method is suitable for industrial production.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a kind of method that means by genetic engineering technique and fermentation strengthen intestinal bacteria L-Phe exocytosis.
Background technology
L-Phe (L-phenylalanine) is a kind of needed by human but the die aromatischen Aminosaeuren with physiologically active that self cannot synthesize, also be a kind of important medicine and edible chemical intermediate, in pharmaceutical industries mainly for the production of antitumor drug and amino acid transfusion preparation, in food service industry, be mainly used in synthetic dipeptide sweetener aspartame, have a good application prospect.The method of producing L-Phe mainly contains four kinds of hydrolysis extraction process, chemical synthesis, enzyme process and fermentation methods, wherein, raw material that microbe fermentation method utilizes is cheap and easy to get, can carry out at normal temperatures and pressures again, is to produce at present the main stream approach of L-Phe both at home and abroad.L-Phe is synthetic by die aromatischen Aminosaeuren metabolic pathway of synthesizing in Bacillus coli cells: glucose synthesizes phosphoenolpyruvic acid and 4-phosphoric acid-erythrose through glycolytic pathway and phosphopentose pathway after entering cell, under the effect of the Arabic heptanone saccharic acid-7-phosphate synthase of 3-deoxidation-D-(AroG), enter shikimic acid pathway, at shikimate kinase (AroK), synthetic chorismic acid under the effect of the enzymes such as shikimate dehydrogenase (YdiB), through enzyme effects such as chorismic acid displacement unit's enzyme and prephenate dehydratases (PheA), generate die aromatischen Aminosaeuren precursor afterwards, pass through again the synthetic L-Phe of effect of tyrosine aminotransferase (TyrB).
The general amino acid accumulation volume of the microorganism strains with acid producing ability filtering out from occurring in nature is limited; Though the mutant strain selecting by special modification can improve the production efficiency of L-Phe, its blindness is large, and workload is heavy, and production cost is high, and mutant strain generally carries auxotrophy, and output further improves and is restricted.By gene clone means, remove metabolic regulation site to promote the synthetic citric acid that has been successfully applied to of object product, lactic acid, the fermentative production of the multiple meta-bolites such as succsinic acid, it is the inexorable trend of L-Phe suitability for industrialized production, but, it is initial that the metabolic regulation of L-Phe is limited to genetic transcription, transcription, translation initiation, translation process, enzyme amount, enzyme work etc. are multilevel, the feedback control of different levels, and because the size of plasmid vector is restricted, be difficult to remove all regulatory sites by the continuous a plurality of genes of overexpression, in addition, intestinal bacteria can accumulate by product---acetic acid during the fermentation, it can limit the growth of thalline and synthesizing of object product, reduce productive rate.In a word, can high yield L-Phe and the cheap engineering strain of fermentation costs have not yet to see report, be difficult to form industrialization.
Summary of the invention
In view of the foregoing defects the prior art has, the object of the present invention is to provide a kind of method that strengthens intestinal bacteria L-Phe exocytosis.The present invention can obviously strengthen intestinal bacteria L-Phe exocytosis level, and L-Phe output is high, and harmful side product acetic acid concentration is low, and fermentation costs is cheap, and technique is simple, suitable for industrial production.
Technical scheme of the present invention is as follows:
A kind of method that strengthens intestinal bacteria L-Phe exocytosis, be the recombinant plasmid transformed host e. coli of overexpression tyrosine aminotransferase TyrB, the Arabic heptanone saccharic acid-7-of outer translocator YddG, the 3-deoxidation-D-of methyl viologen phosphate synthase AroG, chorismate mutase and prephenate dehydratase PheA, shikimate kinase AroK and shikimate dehydrogenase YdiB simultaneously, and produce L-Phe by liquid fermenting.
Its further technical scheme is:
Described recombinant plasmid is TyrB, YddG, AroG, PheA, AroK and the YdiB encoding gene in intestinal bacteria sources to be carried out manually modified to remove its suffered metabolic regulation, and insertion vector builds and forms;
Described liquid fermentation condition is controlled as follows: the initial glucose concn of fermention medium is 3～15g/L, and the glucose concn in fermenting process is 1～4g/L; 35～42 ℃ of leavening temperatures, fermentation pH value 6.5～7.5, air flow 0.5～1.0vvm, dissolved oxygen amount is initial oxygen concentration 10～35%, fermentation time 48～72h.
The natural promoter of described TyrB, YddG, AroG, PheA, AroK and YdiB encoding gene is not replaced by the strong promoter of end meta-bolites feedback repression by other, to strengthen its expression level.
The initial base sequence of described TyrB encoding gene encoder block is replaced by the base sequence shown in SEQ ID NO:1, to remove the binding ability of itself and aporepressor TyrR.
In described AroG aminoacid sequence, the aspartic acid of the 146th is replaced by asparagine, to remove the feedback inhibition of phenylalanine to it.
The R-domain structural domain of described PheA is deleted, to remove the feedback inhibition of phenylalanine to it.
Described strong promoter comprises P
rand/or P
lpromotor
Useful technique effect of the present invention is as follows:
The present invention uses genetic engineering means that TyrB, YddG, AroG, PheA, AroK and the YdiB encoding gene in intestinal bacteria source are carried out manually modified, as introduce strong promoter, delete structural domain, Substitution, amino acid mutation etc., the suffered metabolic regulation of said gene is removed, insertion vector successfully builds and obtains the simultaneously expression plasmid of overexpression TyrB, YddG, AroG, PheA, AroK and YdiB again, and the host e. coli that transforms this plasmid can high yield L-Phe; Wherein, by strengthening the expression level of the required enzyme of L-Phe anabolism path (AroG, PheA, AroK, YdiB and TyrB), can accelerate the synthesis rate of L-Phe, simultaneously, the expression of YddG strengthens the outer transport velocity of born of the same parents that can accelerate L-Phe, reduce intracellular concentration, thereby reduce the feedback inhibition that L-Phe produces metabolism route of synthesis, finally reach the object that improves its metabolic flux and ferment strength; The inventive method can obviously strengthen intestinal bacteria L-Phe exocytosis level, L-Phe output high (can reach 62g/L), and harmful side product acetic acid concentration low (≤3g/L), fermentation costs is cheap, and technique is simple, suitable for industrial production.
Embodiment
By the following examples the present invention is specifically described.
The related test materials of following examples is as follows:
Intestinal bacteria matching DNA extraction is collected center ATCC from intestinal bacteria Escherichia coli str.K-12 substr.W3110(purchased from US mode bacterial classification, and deposit number is ATCC NO:27325); PMD 18T-simple Vector is purchased from precious biotechnology (Dalian) company limited; Restriction enzyme EcoR I, EcoRV, Afl II, Nco I, Kpn I, archaeal dna polymerase, ligase enzyme all flies company purchased from Sai Mo; Plasmid pSY130-14(comprises P
rand P
lpromotor) by the preservation of inventor laboratory, its construction process can be with reference to following bibliographical information: S Sugimoto, M Yabuta, N Kato, T Seki, T Yoshida, H Taguchi (1987) Hyperproduction of phenylalanine by Escherichia coli:application of a temperature-controllable expression vector carrying the repressor-promoter system of bacteriophage lambda.Journal of Biotechnology.5:237 – 253; RNAprep Pure culturing cell/bacterium total RNA extraction reagent box (centrifugal column type is containing DNAase) is purchased from Tian Gen biochemical technology company limited; PrimeScript RT Master Mix Perfect Real Time is purchased from precious biotechnology (Dalian) company limited.
Other raw material and reagent are the commercially available domestic or pure commodity of Import Analysis.
Institute's use plant and instrument is this area routine instrument device.
Following examples seed culture medium used is LB substratum, and it consists of: peptone 10g/L, and yeast powder 5g/L, sodium-chlor 10g/L, all the other compositions are water; Liquid fermentation medium used consists of: K
2hPO
43H
2o 13.57g/L, KH
2pO
44.5g/L, (NH)
4sO
21g/L, MgSO
41g/L, arginine0.1g/L, histidine 0.1g/L, isolecine 0.1g/L, valine 0.1g/L, proline 0.2g/L, p-aminobenzoic acid 0.02g/L, p-hydroxybenzoic0.02g/L, CaCl
20.015g/L, VB
10.075g/L, FeSO
47H
2o 0.1175g/L, Na-Citrate 0.1g/L, trace element solution 1.5ml/L, all the other compositions are that (trace element solution consists of water: Al
2(SO
4)
318H
2o 2g/L, CoSO
47H
2o 0.75g/L, CuSO
45H
2o 2.5g/L, H
3bO
30.5g/L, MnSO
47H
2o 24g/L, Na
2moO
42H
2o3g/L, NiSO
46H
2o 2.5g/L, ZnSO
47H
2o 15g/L, all the other compositions are water).
Embodiment 1 builds the plasmid pR15ABK of overexpression AroG, PheA, AroK and YdiB
The releasing of the Arabic heptanone saccharic acid-7-of 1.1 3-deoxidations-D-phosphate synthase AroG metabolic regulation
The intestinal bacteria matching DNA of take is template, with upstream primer aroG15_F146(base sequence as shown in SEQ ID NO:2) and downstream primer aroG_Hind III _ RV(base sequence as shown in SEQ ID NO:3) carry out pcr amplification and obtain gene fragment A, the intestinal bacteria nucleoid DNA of take is template, with upstream primer aroG_Kpn I _ FW(base sequence as shown in SEQ ID NO:4) and downstream primer aroG15_R146(base sequence as shown in SEQ ID NO:5) carry out pcr amplification and obtain gene fragment B.
Take gene fragment A and B as template, by overlapping PCR(, according to PCR site-directed mutagenesis technique, carry out, reference is as follows: Ho, S.N., H.D.Hunt, R.M.Horton, J.K.Pullen, and L.R.Pease (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction.Gene.77:51-59) amplification object fragment, design of primers is as follows: upstream primer aroG_Sma I _ FW base sequence is as shown in SEQ ID NO:6, downstream primer aroG_Afl II _ EcoR I _ RV base sequence is as shown in SEQ ID NO:7, through Shanghai Sheng Gong biotechnology company limited, check order, the 146th aspartic acid of AroG of determining this Segment A roG15 genes encoding is replaced by asparagine.
By enzyme, cutting ligation is connected to above-mentioned amplified fragments on plasmid pSY130-14, concrete grammar is referring to respective limits restriction endonuclease or ligase enzyme specification sheets, structure obtains plasmid pR15, and this plasmid has been removed the metabolic regulation of phenylalanine to AroG gene, by strong promoter P
rstarting AroG expresses.
The releasing of 1.2 chorismate mutases and prephenate dehydratase PheA metabolic regulation
Take intestinal bacteria matching DNA as template amplification PheA gene catalytic active center 1st～900bp base sequence, delete the R-domain structural domain of being responsible for feedback inhibition, design of primers is as follows: upstream primer pheA_Sma I _ FW base sequence is as shown in SEQ ID NO:8, and downstream primer base sequence pheA_Nco I _ BamH I _ EcoR V _ RV is as shown in SEQ ID NO:9; By enzyme, cut ligation this amplified fragments is connected to above-mentioned plasmid pR15, concrete grammar is referring to respective limits restriction endonuclease or ligase enzyme specification sheets, structure obtains plasmid pR15A, and this plasmid has been removed the metabolic regulation of phenylalanine to AroG and PheA gene, by strong promoter P
rstart AroG and express, by strong promoter P
lstarting PheA expresses.
The releasing of 1.3 shikimate kinase AroK and shikimate dehydrogenase YdiB metabolic regulation
By overlapping PCR(method same 1.1) AroK and YdiB encoding gene are linked together, design of primers is as follows: first pair, upstream primer ydiB_Sma I _ FW base sequence is as shown in SEQ ID NO:10, downstream primer ydiB_aroK_RV base sequence is as shown in SEQ ID NO:11, second pair, upstream primer aroK_ydiB_FV base sequence is as shown in SEQ ID NO:12, and downstream primer aroK_Sma I _ RV base sequence is as shown in SEQ ID NO:13; By enzyme, cut ligation this amplified fragments is connected to above-mentioned plasmid pR15A, be positioned at after PheA encoding gene, concrete grammar is referring to respective limits restriction endonuclease or ligase enzyme specification sheets, structure obtains plasmid pR15ABK, this plasmid has been removed AroG, PheA, AroK and the suffered metabolic regulation of YdiB gene, and makes the natural promoter of said gene by P
rand P
lpromotor is replaced, by strong promoter P
rstart AroG and express, by strong promoter P
lstart the expression of PheA, AroK and YdiB.
The releasing of embodiment 2 tyrosine aminotransferase TyrB metabolic regulations
The intestinal bacteria matching DNA of take carries out pcr amplification as template and obtains tyrB encoding gene, design of primers is as follows: upstream primer tyrB_EcoRV_SD_SB_FW base sequence is as shown in SEQ ID NO:14, downstream primer tyrB_Afl II _ EcoR I _ RV base sequence is as shown in SEQ ID NO:15, wherein, SD sequence A AGGAGGAACAGAC is ribosome bind site, and SB sequence A TGTTCCAGAAGGTCGATG(is as shown in SEQ ID NO:1) be the initial base sequence of tyrB genes encoding frame after replacing.
The acquisition TyrB encoding gene that will increase is connected with pMD 18T-simple Vector, after Shanghai Sheng Gong biotechnology company limited checks order and identifies correctly, by enzyme, cut ligation (concrete grammar is referring to respective limits restriction endonuclease or ligase enzyme specification sheets) and be connected with the plasmid pR15ABK that embodiment 1 builds, tyrB encoding gene is placed in to strong promoter P
rrear, obtains the plasmid of overexpression TyrB, AroG, PheA, AroK and YdiB simultaneously thereby build, by strong promoter P
rstart AroG and tyrB and express, by strong promoter P
lstart the expression of PheA, AroK and YdiB; According to operation steps described in < < molecular cloning experiment guide > >, transform intestinal bacteria Escherichia coli str.K-12 substr.W3110 and carry out liquid fermenting, fermentation ends, recording L-Phe concentration in fermented liquid is 45g/L, and acetic acid concentration is 2.2g/L.
Aforesaid liquid fermentation method for producing is specific as follows:
Seed culture: get inoculation in LB substratum, in 33 ℃ of shaking culture 12h, shaking speed 200rpm, obtains activated seed liquid.
Liquid fermentation and culture: the glucose concn of adjusting liquid fermentation medium with high concentration glucose liquid is 15g/L, with strong aqua, adjust pH value to 6.5～7.5, and to adjust air flow be 0.5vvm, leavening temperature is 35 ℃, with the inoculum size of volume percent 5%, inoculate above-mentioned activated seed liquid, as thalline OD
600=3.0 o'clock, adjusting leavening temperature was 42 ℃, and air flow is 0.8vvm, change mixing speed to control dissolved oxygen amount be initial oxygen concentration 10～35%, by stream, adding high density Glucose Liquid, to control substratum glucose concn be 4g/L, and by stream, adding strong aqua, to control pH value be 6.5～7.5, and 48h ferments.
Above-mentioned L-Phe method for measurement of concentration: adopt C18 reverse chromatograms post, take 7% methyl alcohol as moving phase, detect wavelength 210nm.
Above-mentioned acetic acid concentration measuring method: adopt Shodex SH1011 ion-exchange chromatography at the temperature of 50 ℃, the dilute sulphuric acid of 0.01mol/L of take is moving phase, and flow velocity 0.8ml/min, utilizes differential detector to monitor.
The releasing of outer translocator (methyl viologen efflux pump, the YddG) metabolic regulation of embodiment 3 methyl viologens
The intestinal bacteria matching DNA of take carries out pcr amplification as template and obtains yddG encoding gene, design of primers is as follows: upstream primer yddG_EcoRV_SD_FW base sequence is as shown in SEQ ID NO:16, downstream primer yddG_NcoI_KpnI_RV base sequence is as shown in SEQ ID NO:17, wherein, SD sequence A AGGAGGAACAGAC is ribosome bind site.
The yddG encoding gene that amplification is obtained is connected with pMD 18T-simple Vector, after Shanghai Sheng Gong biotechnology company limited checks order and identifies correctly, by enzyme, cut ligation (concrete grammar is referring to respective limits restriction endonuclease or ligase enzyme specification sheets) and be connected with the plasmid that embodiment 2 builds, yddG encoding gene is placed in to strong promoter P
lrear, build obtain overexpression YddG, TyrB, AroG, PheA, AroK and YdiB simultaneously recombinant expression plasmid, by strong promoter P
rstart AroG and tyrB and express, by strong promoter P
lstart the expression of PheA, AroK, YdiB and YddG.
According to operation steps described in < < molecular cloning experiment guide > >, transform intestinal bacteria Escherichia coli str.K-12 substr.W3110, the structure type L-Phe that is enhanced is produced bacterial strain E.coli W14, using E.coliW14 as fermentation strain, according to method described in embodiment 2, carry out liquid fermenting, fermentation ends, recording L-Phe concentration in fermented liquid is 55g/L, acetic acid concentration is 3g/L, and measuring method is with embodiment 1.
The quantitative fluorescent PCR checking of embodiment 4 TyrB and YddG overexpression
Get E.coli W14 and E.coli W3110 (containing the intestinal bacteria Escherichia coli str.K-12substr.W3110 that transforms plasmid pR15ABK) and be inoculated in respectively LB substratum, in 33 ℃ of overnight shakings, cultivate, then the inoculum size of volume percent 5% of take is seeded to liquid fermentation medium (adding the glucose that final concentration is 5g/L), in 35 ℃ of shaking culture 9h, transfer again 38 ℃ of cultivations to, and in cultivating 11h and 15h sampling, adopt RNAprep Pure culturing cell/bacterium total RNA extraction reagent box (centrifugal column type is containing DNAase) to extract total RNA, concrete operation step is referring to test kit working instructions, and adopt PrimeScriptRT Master Mix Perfect Real Time at CFX96 Bio-Rad Real-Time PCR Detection System(Bio-Rad) carry out quantitative fluorescent PCR, using ihfB as reference gene, with E.coli W3110 bacterial strain in contrast, in CFX96 Bio-Rad Real-Time PCR Detection System(Bio-Rad) respectively to tyrB, yddG and ihfB gene carry out fluorescent quantitative PCR, tyrB, the amplification upstream and downstream primer base sequence (TyrBa of yddG and ihfB gene, TyrBb, YddGa, YddGb, ihfBa, ihfBb) respectively as SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, shown in SEQ ID NO:23, amplification condition is as follows: 95 ℃ of 30s of denaturation, then 95 ℃ of 5s, 60 ℃ of 35s circulate 40 times, and adopt gradient dilution method to obtain typical curve, yddG:y=-3.286x+11.895(E=101.5%R
2=0.981), tyrB:y=-3.334x+15.335(E=99.5%R
2=0.988), ihfB:y=-3.364+21.121(E=98.3.1%R
2=0.988), adopt 2
-Δ Δ C t(reference is as follows: Livak K, Shmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the2 for method
-Δ Δ C tmethod.Methods25:402 – 408) calculate the expression level of tyrB and yddG, compare with E.coli W3110, in E.coli W14 bacterial strain, the expression level of tyrB and yddG is increased to respectively 4.85 and 5.17 at cultivation 11h, 15h is increased to respectively 12.57 and 5.97, proves E.coli W14 bacterial strain overexpression tyrB and yddG simultaneously.Above data analysis adopts CFX Manager 3.0(Bio-Rad) carry out.
The overexpression of embodiment 5 yddG is in born of the same parents and the impact of the outer L-Phe concentration of born of the same parents
The outer L-Phe method for measurement of concentration of born of the same parents in born of the same parents in M9 substratum: by E.coli W14 and E.coliW3110(with embodiment 4, control strain) be inoculated in respectively LB substratum, in 200rpm, cultivate after 9h, be warming up to 38 ℃ and continue to cultivate 2h, take out stroke-physiological saline solution (0.9%NaCl) washed twice for thalline, then use M9 substratum (first to prepare 5 * M9 salts solution: Na
2pO
47H
2o 12.8g, KH
2pO
43.0g, NaCl0.5g, NH
4cl 1.0g, sterilizing, gets 5 * M9 salts solution 200ml during use, add the MgSO of sterilized 1mol/L
42ml, the CaCl of 1mol/L
20.1ml, 20% glucose solution 20ml, is settled to 1000ml with sterilizing distilled water) adjustment OD
600to 6, in 38 ℃ of cultivations, L-Phe concentration outside born of the same parents in 1h, 2h, 3h and 8h sampling and measuring born of the same parents.In born of the same parents, L-Phe measuring method is as follows: get appropriate fermented liquid and adjust OD
600to 3(, being equivalent to thalline quantity is 1～2 * 10
9cFU/ml), centrifugal collection 1mL thalline, washing twice, adds 250 μ L 35% perchloric acid in vibrator thermal agitation 5min, adds 280 μ L 2.7mol/L Na
2cO
3neutralization, the centrifugal cell debris of removing, as liquid phase analysis, L-Phe method for measurement of concentration is 1 μ m with embodiment 1(hypothesis Bacillus coli cells volume
3).Experimental result is referring to table 1 and table 2.
The outer L-Phe change in concentration of born of the same parents in table 1 M9 substratum
L-Phe change in concentration in born of the same parents in table 2 M9 substratum
From table 1 and table 2 data, cultivate 8h in M9 substratum after, in E.coli W14 born of the same parents, L-Phe concentration is starkly lower than control strain, and the outer L-Phe concentration of born of the same parents also increases compared with control strain simultaneously, proves that the overexpression of yddG has strengthened the exocytosis of L-Phe.
The 15L ferment tank of embodiment 5 E.coli W14 is cultivated
Preparation 6L liquid fermentation medium is loaded in 15L fermentor tank, 121 ℃ of sterilizing 20min, adjustment initial glucose concentration is 15g/L, add strong aqua to adjust pH value to 7.5, adjustment air flow is 0.5vvm, fermentation jar temperature is 35 ℃, and the activated seed liquid of cultivating 12h in LB substratum is inoculated in fermentor tank, cultivates OD
600it is 3 o'clock, adjust leavening temperature to 42 ℃, open large ventilation to 0.8vvm, change mixing speed control dissolved oxygen amount is 10～35% simultaneously, it is 1～4g/L that the glucose solution that the data rate stream of 2g/L/h of take adds 600g/L is controlled glucose concn, by stream, adding strong aqua control pH value is 6.5～7.5, and 48h altogether ferments.Fermentation ends, measuring L-Phe concentration in fermented liquid is 46g/L, and acetic acid concentration is 2.4g/L, and dry cell weight reaches 40g/L, and measuring method is with embodiment 2.
The 30L ferment tank of embodiment 6 E.coli W14 is cultivated
Preparation 20L liquid fermentation medium is loaded in 30L fermentor tank, 115 ℃ of sterilizing 30min, adjustment initial glucose concentration is 3g/L, add strong aqua to adjust pH value to 6.5, adjustment air flow is 0.8vvm, fermentation jar temperature is 42 ℃, and the activated seed liquid of cultivating 12h in LB substratum is inoculated in fermentor tank, is cultured to OD
600it is 15 o'clock, adjust leavening temperature to 38 ℃, open large ventilation to 1.0vvm, change mixing speed control dissolved oxygen amount is 10～35% simultaneously, the glucose solution that adds 600g/L with the data rate stream of 2g/L/h is controlled glucose concn between 1～4g/L, adds strong aqua control pH between 6.5～7.5 by stream, and 65h altogether ferments.Fermentation ends, measuring phenyl-alanine concentration in fermented liquid is 62g/L, and acetic acid is concentration 2.7g/L, and dry cell weight reaches 30g/L, and measuring method is with embodiment 2.
The above be only the preferred embodiment of the present invention, the invention is not restricted to above embodiment.Be appreciated that other improvement and variation that those skilled in the art directly derive without departing from the spirit and concept in the present invention or associate, within all should thinking and being included in protection scope of the present invention.
Claims (1)
1. a method that strengthens intestinal bacteria L-Phe exocytosis, it is characterized in that: the recombinant plasmid transformed host e. coli of overexpression tyrosine aminotransferase TyrB, the Arabic heptanone saccharic acid-7-of outer translocator YddG, the 3-deoxidation-D-of methyl viologen phosphate synthase AroG, chorismate mutase and prephenate dehydratase PheA, shikimate kinase AroK and shikimate dehydrogenase YdiB simultaneously, and produce L-Phe by liquid fermenting; Described liquid fermentation condition is controlled as follows: the initial glucose concn of fermention medium is 3 ~ 15g/L, glucose concn in fermenting process is 1 ~ 4g/L, 35 ~ 42 ℃ of leavening temperatures, fermentation pH value 6.5 ~ 7.5, air flow 0.5 ~ 1.0vvm, dissolved oxygen amount is 10 ~ 35% of initial oxygen concentration, fermentation time 48 ~ 72h;
Described recombinant plasmid is TyrB, YddG, AroG, PheA, AroK and the YdiB encoding gene in intestinal bacteria sources to be carried out manually modified to remove its suffered metabolic regulation, and insertion vector builds and forms;
The natural promoter of described TyrB, YddG, AroG, PheA, AroK and YdiB encoding gene is not replaced by the strong promoter of end meta-bolites feedback repression by other, to strengthen its expression level, described strong promoter comprises P
r-and/or P
lpromotor;
The initial base sequence of described TyrB encoding gene encoder block is replaced by the base sequence shown in SEQ ID NO:1, to remove the binding ability of itself and aporepressor TyrR;
In described AroG aminoacid sequence, the aspartic acid of the 146th is replaced by asparagine, to remove the feedback inhibition of phenylalanine to it;
The R-domain structural domain of described PheA is deleted, to remove the feedback inhibition of phenylalanine to it.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310074890.6A CN103146773B (en) | 2013-03-08 | 2013-03-08 | Method for enhancing L-phenylalanine exocytosis of escherichia coli |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310074890.6A CN103146773B (en) | 2013-03-08 | 2013-03-08 | Method for enhancing L-phenylalanine exocytosis of escherichia coli |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103146773A CN103146773A (en) | 2013-06-12 |
| CN103146773B true CN103146773B (en) | 2014-08-27 |
Family
ID=48545091
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310074890.6A Active CN103146773B (en) | 2013-03-08 | 2013-03-08 | Method for enhancing L-phenylalanine exocytosis of escherichia coli |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103146773B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104745500B (en) * | 2013-12-31 | 2017-10-20 | 沙县鑫创生物科技有限公司 | One plant production L phenylalanines bacterial strain and its application |
| EP3760723A4 (en) * | 2018-03-02 | 2021-12-15 | Japan Science and Technology Agency | Method for producing 4-aminocinnamic acid, and vector and host cell used in same |
| CN116144560A (en) * | 2022-09-28 | 2023-05-23 | 天津科技大学 | Phenylalanine escherichia coli production strain and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0336452A1 (en) * | 1983-02-17 | 1989-10-11 | Kyowa Hakko Kogyo Co., Ltd. | Process for preparing L-phenylalanine |
| 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 |
| CN102010847A (en) * | 2010-03-15 | 2011-04-13 | 江南大学 | Antiphagin L-phenylalanine producing strain as well as breeding method and application thereof |
| 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 |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61260892A (en) * | 1985-05-14 | 1986-11-19 | Kyowa Hakko Kogyo Co Ltd | Production of l-phenylalanine |
| JPH05123178A (en) * | 1991-11-01 | 1993-05-21 | Ajinomoto Co Inc | Production of l-phenylalanine |
-
2013
- 2013-03-08 CN CN201310074890.6A patent/CN103146773B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0336452A1 (en) * | 1983-02-17 | 1989-10-11 | Kyowa Hakko Kogyo Co., Ltd. | Process for preparing L-phenylalanine |
| 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 |
| CN102010847A (en) * | 2010-03-15 | 2011-04-13 | 江南大学 | Antiphagin L-phenylalanine producing strain as well as breeding method and application thereof |
| 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 |
Non-Patent Citations (10)
| Title |
|---|
| Chorismate Mutase-Prephenate Dehydratase from Escherichia coli;Sheng Zhang et.al.,;《The Journal of biological chemistry》;19980313;第273卷(第11期);第6248页左栏第1段 * |
| Ji Yang et.al.,.Molecular analysis of tyrosine- and phenylalanine-mediated repression of the tyrB promoter by the TyrR protein of Escherichia coli.《Molecular Microbiology》.2002,第45卷(第5期),第1407页摘要. |
| JP昭61-260892A 1986.11.19 |
| JP特开平5-123178A 1993.05.21 |
| Molecular analysis of tyrosine- and phenylalanine-mediated repression of the tyrB promoter by the TyrR protein of Escherichia coli;Ji Yang et.al.,;《Molecular Microbiology》;20021231;第45卷(第5期);第1407页摘要 * |
| Mutational Analysis of the Feedback Sites of Phenylalanine-Sensitive 3-Deoxy-D-arabino-Heptulosonate-7-Phosphate Synthase of Escherichia coli;Y Kikuchi et.al.,;《Applied and environmental microbiology》;19970228;第63卷(第2期);第761页右栏第1段，表1 * |
| Sheng Zhang et.al.,.Chorismate Mutase-Prephenate Dehydratase from Escherichia coli.《The Journal of biological chemistry》.1998,第273卷(第11期),第6248页左栏第1段. |
| Y Kikuchi et.al.,.Mutational Analysis of the Feedback Sites of Phenylalanine-Sensitive 3-Deoxy-D-arabino-Heptulosonate-7-Phosphate Synthase of Escherichia coli.《Applied and environmental microbiology》.1997,第63卷(第2期),第761页右栏第1段，表1. |
| 大肠杆菌苯丙氨酸代谢途径的分子改造;马蓉;《中国优秀硕士学位论文全文数据库 基础科学辑》;20130215(第2期);第V、7、9、31、45-46页 * |
| 马蓉.大肠杆菌苯丙氨酸代谢途径的分子改造.《中国优秀硕士学位论文全文数据库 基础科学辑》.2013,(第2期),第V、7、9、31、45-46页. |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103146773A (en) | 2013-06-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108467860B (en) | Method for high yield of gamma-aminobutyric acid | |
| CN109988799B (en) | Application of glycerol-2-alpha-glucosylation enzyme in preparation of 2-alpha-glycerol glucoside | |
| CN107603937B (en) | Recombinant escherichia coli for expressing lysine aminopeptidase and construction method thereof | |
| CN111172127A (en) | Application of sucrose phosphorylase in preparation of glycerol glucoside | |
| CN103146773B (en) | Method for enhancing L-phenylalanine exocytosis of escherichia coli | |
| CN104745520B (en) | A kind of strain excellent of high yield L phenylalanines and its application | |
| CN104404075A (en) | Method for catalyzing to generate L-theanine by using recombinant Bacillus subtilis secreted gamma-glutamyltranspeptidase | |
| CN107916283B (en) | A kind of production technology of niacinamide | |
| CN111019960B (en) | Method for preparing spermidine by enzyme method | |
| CN104046586B (en) | One strain gene engineering bacterium and the application in producing (2R, 3R)-2,3-butanediol thereof | |
| CN103131724B (en) | Efficient engineering bacteria recombinant protein expression method and applications thereof | |
| CN110184229B (en) | Method for improving efficiency of synthesizing N-acetylglucosamine by recombinant bacillus subtilis | |
| CN113337495B (en) | Method for improving sialic acid yield and application | |
| CN101679936B (en) | Method for producing glucuronic acid by glucuronic acid fermentation | |
| CN108410789B (en) | Microorganism for producing poly-gamma-glutamic acid and construction method and application thereof | |
| CN112251428B (en) | Glutamic acid decarboxylase mutant and application thereof in production of gamma-aminobutyric acid | |
| CN110257313B (en) | Engineering bacterium of bacillus amyloliquefaciens for producing spermidine | |
| CN105255934A (en) | Strategy for efficiently coproducing alpha-aminobutyric acid and gluconic acid | |
| CN108048500A (en) | The biological synthesis method of Beta-alanine | |
| CN111518711B (en) | Enterobacter strain and application thereof in coproduction of microbial exopolysaccharide and 2,3-butanediol | |
| CN105950687A (en) | Fucose catalytic synthesis method using self-induction culture medium and whole cells | |
| CN109266707A (en) | A method of preparing poly sialic acid | |
| JP5946080B2 (en) | Method for producing plastic raw materials and related substances in cyanobacteria | |
| CN1219071C (en) | Method for producing yeast extracellular trehalose by two step fermentation method | |
| CN110804602B (en) | L-aspartic acid beta-decarboxylase mutant and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CP02 | Change in the address of a patent holder |
Address after: 214016 Jiangsu city of Wuxi province Tong Road No. 898 South 7 Patentee after: Jiangnan University Address before: No. 1800 road 214122 Jiangsu Lihu Binhu District City of Wuxi Province Patentee before: Jiangnan University |
|
| CP02 | Change in the address of a patent holder |