CN107446870A - A kind of preparation method of high yield thick grass mutant polynucleotides - Google Patents

A kind of preparation method of high yield thick grass mutant polynucleotides Download PDF

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CN107446870A
CN107446870A CN201710395727.8A CN201710395727A CN107446870A CN 107446870 A CN107446870 A CN 107446870A CN 201710395727 A CN201710395727 A CN 201710395727A CN 107446870 A CN107446870 A CN 107446870A
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seq
sequence
amino acid
base sequence
sequence table
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CN107446870B (en
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唐双焱
梁朝宁
李恒
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Institute of Microbiology of CAS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids

Abstract

The present invention can not realize the present situation of high flux screening for current thick grass acid yield, provide a kind of gene lacks functionality Escherichia coli of high yield shikimic acid, characterized in that, gene sucC or agp or nanS or pphB or the protein delation function expressed by bglF or promoter of sdhCDAB sucABCD operon or and；Described gene sucC has the base sequence described in SEQ ID NO.1 in sequence table；Described gene agp has the base sequence described in SEQ ID NO.2 in sequence table；Described gene nanS has the base sequence described in SEQ ID NO.3 in sequence table；Described gene pphB has the base sequence described in SEQ ID NO.4 in sequence table；Described gene bglF has SEQ ID NO. in sequence tableDescribed base sequence；Described gene promoter of sdhCDAB sucABCD operon have SEQ ID NO. in sequence tableDescribed base sequence；Described gene and has the amino acids basic sequence described in SEQ ID NO.7 in sequence table.The present invention realizes the high yield of thick grass acid yield.

Description

A kind of preparation method of high yield thick grass mutant polynucleotides
Technical field
The present invention relates to the technical field of shikimic acid screening technique.
Background technology
Shikimic acid (shikimic acid, SA) is synthetic aromatic amino acid, alkaloid and many high added value bios The important intermediate of product.In recent years, bird flu epidemic situation spreads in the world, and shikimic acid is as unique effectively prevention and treatment The critical materials of bird flu medicine Oseltamivir Phosphate (trade name " Tamiflu "), therefore receive much concern.In addition, shikimic acid is also It can be applied to synthesizing antineoplastic medicament two and dislike mycin, aldoketonutase inhibitor.Compared to traditional from the plant fruit such as anise The method and the method for chemical synthesis extracted in reality, Production by Microorganism Fermentation shikimic acid have unrivaled advantage, such as raw Produce the cycle is short, environmental hazard is small, cost is low etc..Wherein, using classical engineered strain Escherichia coli as host synthesize shikimic acid as One of conventional Microbe synthesis mode.
As a kind of micromolecular compound, although shikimic acid has the huge market demand, the micro- of high yield is had no at present Biological fermentation process produces shikimic acid.
The content of the invention
The technical problem to be solved in the present invention is can not to realize high yield present situation for current thick grass acid yield.
First, the invention provides a kind of gene lacks functionality Escherichia coli of high yield shikimic acid, gene sucC or agp, Or nanS or pphB or the albumen expressed by bglF or promoter of sdhCDAB-sucABCD operon or gnd lack Lose function；
Described gene sucC has the base sequence described in SEQ ID NO.1 in sequence table；
Described gene agp has the base sequence described in SEQ ID NO.2 in sequence table；
Described gene nanS has the base sequence described in SEQ ID NO.3 in sequence table；
Described gene pphB has the base sequence described in SEQ ID NO.4 in sequence table；
Described gene bglF has SEQ ID NO. in sequence tableDescribed base sequence；
Described gene promoter of sdhCDAB-sucABCD operon have SEQ ID NO. in sequence tableInstitute The base sequence stated；
Described gene gnd has the base sequence described in SEQ ID NO.7 in sequence table.
Secondly, present invention also offers a kind of method for producing shikimic acid：By paroGfbrElectricity is converted in gene lacks functionality Coli strain DHIn α Δs aroA, be applied to afterwards card receive mycin flat board (0 μ g/ml), obtained strain is connect with 0.01OD Kind is into LB culture mediums, 20rpm, 12h is cultivated under the conditions of 37 DEG C, utilize thick grass acid yield in HPLC detection zymotic fluids.
Further, described paroGfbrFor：
With the base sequence in sequence table shown in SEQ ID NO.8 or the amino acid sequence shown in SEQ ID NO.28 Protein, or
With the base sequence in sequence table shown in SEQ ID NO.9 or the amino acid sequence shown in SEQ ID NO.29 Protein, or
With the base sequence in sequence table shown in SEQ ID NO.10 or the amino acid sequence shown in SEQ ID NO.30 Protein, or
With the base sequence in sequence table shown in SEQ ID NO.11 or the amino acid sequence shown in SEQ ID NO.31 Protein, or
With the base sequence in sequence table shown in SEQ ID NO.12 or the amino acid sequence shown in SEQ ID NO.32 Protein, or
With the base sequence in sequence table shown in SEQ ID NO.13 or the amino acid sequence shown in SEQ ID NO.33 Protein, or
With the base sequence in sequence table shown in SEQ ID NO.14 or the amino acid sequence shown in SEQ ID NO.34 Protein, or
With SEQ ID NO.1 in sequence tableShown base sequence or SEQ ID NO.3Shown amino acid sequence Protein, or
With SEQ ID NO.1 in sequence tableShown base sequence or SEQ ID NO.3Shown amino acid sequence Protein, or
With the base sequence in sequence table shown in SEQ ID NO.17 or the amino acid sequence shown in SEQ ID NO.37 Protein, or
With the base sequence in sequence table shown in SEQ ID NO.18 or the amino acid sequence shown in SEQ ID NO.38 Protein, or
With the base sequence in sequence table shown in SEQ ID NO.19 or the amino acid sequence shown in SEQ ID NO.39 Protein, or
With the base sequence in sequence table shown in SEQ ID NO.20 or the amino acid sequence shown in SEQ ID NO.40 Protein.Or
With the base sequence in sequence table shown in SEQ ID NO.21 or the amino acid sequence shown in SEQ ID NO.41 Protein.Or
With the base sequence in sequence table shown in SEQ ID NO.22 or the amino acid sequence shown in SEQ ID NO.42 Protein.Or
With the base sequence in sequence table shown in SEQ ID NO.23 or the amino acid sequence shown in SEQ ID NO.43 Protein.Or
With the base sequence in sequence table shown in SEQ ID NO.24 or the amino acid sequence shown in SEQ ID NO.44 Protein.Or
With SEQ ID NO.2 in sequence tableShown base sequence or SEQ ID NO.4Shown amino acid sequence Protein.Or
With SEQ ID NO.2 in sequence tableShown base sequence or SEQ ID NO.4Shown amino acid sequence Protein.Or
With the base sequence in sequence table shown in SEQ ID NO.27 or the amino acid sequence shown in SEQ ID NO.47 Protein.
Brief description of the drawings
The uricase modulin HucR vector construction figures of Fig. 1 random mutations transformation.
Fig. 2 mutant HucR-4 relatively schemes with wild type shikimic acid induction ratio.
Embodiment
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
1 structure mutated library
Using pSH plasmids as template, in 0.2mM dATP and dGTP, 1.0mM dCTP and dTTP,mM MgCl2, 7μM MnCl2Sudden change conditions under.With-atggctagcatgttgaagaaaaaag-3’(forward)and- Gtgctcgagtacctttccaacaccagc-3 ' (reverse) is that primer enters performing PCR amplification, the PCR primer being mutated.Will The PCR primer of mutation NdeI and NheI double digestions, digestion products connect with the pSH carriers through identical digestion respectively, connection production Thing converts to DH respectivelyIn α competent cell, mutated library is obtained.
2 prepare the mutant HucR-4 that can be induced by shikimic acid
Added to mutant library containing 100mg/L ammonia benzyl mycins and with the addition of the LB liquid medium of 20mM shikimic acids In, 20rpm, 12h is cultivated under the conditions of 37 DEG C.Stronger cell is expressed to reporter gene RFP with flow cytometer and carries out richness Collection, obtain the mutant HucR-4 that can be induced by shikimic acid.
3 mutant HucR-4 and wild type HucR contrasts
By mutant HucR-4 and wild type HucR 0mM, 7.mM、10mM、17.A series of concentration of mM, 20mM it is big Oxalic acid is induced, 20rpm, 12h is cultivated under the conditions of 37 DEG C, detect red fluorescent protein, the results showed that wild type HucR is several Do not induced by shikimic acid, and mutant HucR-4 can be induced up to 14 times by additional shikimic acid.
Thick grass acid content and reporter gene RFP expression are proportionate, therefore this detecting system can be utilized to characterize Thick grass acid content, build shikimic acid high-throughput screening method.
4 establish transposons radom insertion library
By Tn10 Transposon plasmids (1 μ g) electricity conversion to Escherichia coli DHIn α Δs aroA competent cell, 37 DEG C multiple Revive after 1h, resuscitation fluid is applied to containing 1mM IPTG and0mg/L cards receive mycin LB flat boards on, obtain transposons radom insertion Library.
Detect thick grass acid yield
By paroGfbrElectricity is converted to coli strain DHα Δ aroA, be applied to afterwards card receive mycin flat board (0μg/ Ml), obtained strain is inoculated into LB culture mediums with 0.01OD, 20rpm, 12h is cultivated under the conditions of 37 DEG C, detect thick grass Acid yield.
By mutant HucR-4 and paroGfbrElectricity conversion is applied to containing 100mg/L ammonia benzyls to transposons radom insertion library On the LB flat boards of mycin and 30mg/L chloramphenicol, it is next that red deeper single bacterium colony progress on flat board is selected after 37 DEG C of culture 18h Walk secondary screening.It is inoculated into what is selected in test tube, 20rpm, 12h is cultivated under the conditions of 37 DEG C, survey RFP values, selection fluorescent value is most High mutant detection thick grass acid yield.
1ml zymotic fluids are taken, centrifugation (12,000 × g) takes supernatant after 1 minute.HPLC is carried out after 0.22 μm of membrane filtration Test.
HPLC conditions are：Equipment Shimadzu LC-20A HPLC system, liquid-phase chromatographic column are Bio-Rad Aminex HPX-87H chromatographic columns (10 μm, 7.8 × 300mm), detector are SPD-M20A PDADs, and Detection wavelength is 210nm.Separation condition is：Mobile phase：MM sulfuric acid solution, flow velocity 0.4Ml/ minutes.
The appearance time of shikimic acid is about 1.7 minute.With the shikimic acid sample introduction of series concentration, draw thick grass acid concentration and HPLC goes out the standard curve of peak area, the final thick grass acid concentration for calculating test sample.
It is determined that knock out gene
Finally determine that the insertion of multiple genes inactivates and cause shikimic acid output increased, wherein 4 gene/gene clusters are first It was found that：SucC, agp, hanS, pphB, bglF, promoter of sdhCDAB-sucABCD operon, gnd.Shikimic acid Yield is respectively increased to 1.4、18.9、20.7、14.8、19.8、19.0、21.0mg/L。
7 gene knockouts are verified
Gene/gene cluster sucC, agp, nanS, pphB, bglF, promoter of sdhCDAB-sucABCD Operon, gnd are knocked out with authentication function in three kinds of different types of escherichia coli hosts respectively.The large intestine bar of three types Bacterium host is respectively：DHα、BW2113、JM109。
Conversion there is into pKD4Treat knock-out bacterial strain be inoculated with, 30 DEG C of overnight incubations；Next day 1:0 is seeded to 100ml LB cultures Base (ampicillin concentration 100mg/ml), 30 DEG C of cultures to D00=0.3When, L-arabinose is added to 1mM, induction 1h(D00 no more than 0.), make pKD4On tri- albumen of Exo, Bet and Gam give full expression to.Precooling 10min on ice, 4000r/min, 4 DEG C of centrifugation 10min, abandons culture medium；With 10% glycerine centrifuge washing of precooling 3 times, 100 times of senses into 1ml are concentrated By state cell, every μ l of pipe 100.By the card that homology arm primer expands receive mycin resistant gene fragment about 800ng add competence it is thin Born of the same parents, mix, be transferred in 0.2cm electric shock cups, make electricity conversion with Bio-Rad electric shock instruments.Electric shock condition：200 Ψ, 2μ F, electric shock electricity Press 2.3kV, electric shock the time be 4~Ms, is rapidly added 1ml LB after electric shock, and 20r/min, 37 DEG C of culture 1h, is applied to card afterwards Receive mycin flat board (0μg/ml)；Chloramphenicol resistance is received after 12h with the card that grows of PCR methods identification to clone.
DHα, JM109 are purchased from precious bioengineering (Dalian) Co., Ltd, knock out aroA genes afterwards, and knockout technique is same as above. BW2113 Δ aroA come from keio collection (Baba T, Ara T, Hasegawa M, et al.Construction Of Escherichia coli K-12 in-frame, single-gene knockout mutants：the Keio Collection. [J] .Molecular Systems Biology, 2006,2 (1)：2006.0008-2006.0008.).
Wild-type strain DHAroG is overexpressed in α Δs aroAfbrYield is 7.8mg/L afterwards, and base is knocked out on the basis of this bacterium It is big after cause/gene cluster sucC, agp, nanS, pphB, bglF, promoter of sdhCDAB-sucABCD operon, gnd Oxalate Production is respectively increased to 14.9,14.1,22.12.0,14.8,19.8,23.mg/L。
Wild-type strain BW2AroG is overexpressed in 113 Δ aroAfbrYield is 7. afterwardsMg/L, knocked out on the basis of this bacterium After gene/gene cluster sucC, agp, nanS, pphB, bglF, promoter of sdhCDAB-sucABCD operon, gnd, Thick grass acid yield is respectively increased to 1.9,1.0,24.1,13.2,1.1,20.0,24.mg/L。
AroG is overexpressed in wild-type strain JM109 Δs aroAfbrYield is 7.1mg/L afterwards, and base is knocked out on the basis of this bacterium It is big after cause/gene cluster sucC, agp, nanS, pphB, bglF, promoter of sdhCDAB-sucABCD operon, gnd Oxalate Production is respectively increased to 13.8,12.8,21.8,11.9,12.7,1.9,20.1, mg/L.
8 enzyme AroGfbrLibraries of random mutants is built
(1) primer
Primer I are-tccgaattcatgaattatcagaacgacga-3’；
Primer II are-agactgcagttacccgcgacgcgcttttac-3’。
(2) fallibility PCR reaction systems
Contain 1 × Taq DNA polymerase buffers liquid, 0.2mM dATP and dGTP, 1mM dCTP and dTTP in 20 μ L systems,0.3~0.8mM Mn2+, 1mM Primer I and Primer II, template DNA is plasmid paroGfbrAbout 10ng。
(3) amplification and identification
94 DEG C,min；94 DEG C, 1min；DEG C, 1.min；72 DEG C, 1.min；30 circulations；72 DEG C, 10min, use matter Measure the agarose gel electrophoresis that fraction is 0.8% and identify fallibility PCR primer, using glue reclaim kit gel extraction PCR primer, In -20 DEG C of preservations.
(4) library construction
By above-mentioned fallibility PCR primer through glue reclaim after purification, through EcoR I-Pst I double digestions, the load with same double digestion Body paroGfbrIt is connected, recon introduces DHα, extraction plasmid obtain mutant library.
Mutant HucR-4 and mutant library electricity are converted to Escherichia coli DHIn α Δs aroA competent cell, apply It is distributed in screening flat board (tryptone.0gL-1, yeast extract 3.0gL-1, sodium chloride 10.0gL-1,100mg/L ammonia benzyl Mycin, the LB flat boards of 30mg/L chloramphenicol, agar powder 10.0gL-1), observe growth and the discoloration of the single bacterium colony of flat board The deeper red single bacterium of picking color, which is inoculated in LB fluid nutrient mediums, cultivates, and collects thalline afterwards and carries out HPLC secondary screening mirror It is fixed.Tested by the ability HPLC that several plants of E. coli mutant strains are produced with shikimic acid, test shows mutant AroGfbrM1 Improved to M20 thick grass acid yield compared with wild-type e. coli yield (7.8mg/L)
9AroGfbrDAPH synthase activities detect
By mutant AroGfbrWith-atggctagcatgaattatcagaacgacgat-3’(forward)and- Ctcgaattcttacccgcgacgcgctttta-3 ' (reverse) is that primer enters performing PCR amplification, the PCR primer being mutated. It is used to NheI and EcoRI double digestions, digestion products respectively with the pET28a carriers (being purchased from Promega companies) through identical digestion Plasmid pET28a-aroG M2 are obtained with 4 DEG C of connections of T4 ligases (being purchased from precious biotech firm).
Above-mentioned plasmid is transferred in BL21 (DE3) Host Strains, BL21 (DE3)/pET28a-aroG of acquisition.Single bacterium colony connects Kind is (final concentration of to kanamycins is contained0 μ g/ml) LB fluid nutrient mediums in, 37 DEG C culture 12h, collect zymotic fluid, will send out Zymotic fluid is forwarded in the fresh LB fluid nutrient mediums of 100ml according to the inoculum concentration of 1% (volumn concentration), and 37 DEG C of cultures are extremelyReach 0., then sterile IPTG is added in the medium, the final concentration of 1mM of IPTG in the medium is entered at 30 DEG C Row induction fermentation.Terminate fermentation after 12h, zymotic fluid 4000g centrifugations 10min is collected into thalline.
Thalline is resuspended in0ml concentration is0mM combination buffer (Tris- hydrochloric acid, pH8,300mM NaCl, 10mM Imidazoles), ultrasonication (200W, work 3s pause 3s, work 100 times), centrifugation (1700g, 4 DEG C, 30min) to remove cell broken Piece, collect supernatant.By above-mentioned supernatant after 0.22 μm of membrane filtration, purified using nickel post, the condition of purifying is： Wash buffer：0mM pH8 Tris-HCl, 300mM NaCl, 20mM imidazoles；Elution buffer：0mM pH8 Tris- HCl, 300mM NaCl, 20mM imidazoles.Efflux is collected, (dialyzate is through dialysis treatment0mM 1,3- are double ((trihydroxy methyl) Methylamino) Propane buffer (pH 7.0), the molecular cut off 8000-14000 of bag filter) after, obtain purifying enzyme liquid.
Difference (PEP, εs of the DAHP synthesis enzymatic determinations two kinds of reaction substrate PEP and E4P of enzyme activity foundation in 232nm absorbance =2800, E4P, ε=41) be measured.All reagents will be with just can be used after 0.22 μm of membrane filtration.Reaction is in 10mM Double ((trihydroxy methyl) methylamino) Propane buffers (pH 7.0) of 1,3- in carry out, final concentration of the 2 of substrate PEP and E4P - 200mM, cumulative volume 1ml, 2DEG C preheatingThe DAHP synzyme initial actions of 2ul after purification are added after min.Utilize ELIASA (SynergyMx, biotek, USA) detects OD value changes situation (2 of the reaction solution in 232nm℃).One enzyme-activity unit definition Under the conditions of standard reaction, 1min can generate zymoprotein amount needed for 1 μm of ol DAHP.
Purifying enzyme liquid (mutation) Rate activity is：
The above results show that mutant generation DAHP ability all increases.

Claims (3)

  1. A kind of 1. gene lacks functionality Escherichia coli of high yield shikimic acid, it is characterised in that gene sucC or agp or nanS, Or pphB or the protein delation function expressed by bglF or promoter of sdhCDAB-sucABCD operon or gnd；
    Described gene sucC has the base sequence described in SEQ ID NO.1 in sequence table；
    Described gene agp has the base sequence described in SEQ ID NO.2 in sequence table；
    Described gene nanS has the base sequence described in SEQ ID NO.3 in sequence table；
    Described gene pphB has the base sequence described in SEQ ID NO.4 in sequence table；
    Described gene bglF has in sequence tableDescribed base sequence；
    Described gene promoter of sdhCDAB-sucABCD operon have in sequence tableDescribed Base sequence；
    Described gene gnd has the amino acids basic sequence described in SEQ ID NO.7 in sequence table.
  2. A kind of 2. method for producing shikimic acid, it is characterised in that including：
    By paroGfbrElectricity is converted in gene lacks functionality Escherichia coli, and obtained strain is applied into card receives mycin flat board It is inoculated into 0.01OD in LB culture mediums,12h is cultivated under the conditions of 37 DEG C.
  3. 3. according to the method for claim 2, it is characterised in that described paroGfbrFor：
    Albumen with the base sequence in sequence table shown in SEQ ID NO.8 or the amino acid sequence shown in SEQ ID NO.28 Matter, or
    Albumen with the base sequence in sequence table shown in SEQ ID NO.9 or the amino acid sequence shown in SEQ ID NO.29 Matter, or
    Egg with the base sequence in sequence table shown in SEQ ID NO.10 or the amino acid sequence shown in SEQ ID NO.30 White matter, or
    Egg with the base sequence in sequence table shown in SEQ ID NO.11 or the amino acid sequence shown in SEQ ID NO.31 White matter, or
    Egg with the base sequence in sequence table shown in SEQ ID NO.12 or the amino acid sequence shown in SEQ ID NO.32 White matter, or
    Egg with the base sequence in sequence table shown in SEQ ID NO.13 or the amino acid sequence shown in SEQ ID NO.33 White matter, or
    Egg with the base sequence in sequence table shown in SEQ ID NO.14 or the amino acid sequence shown in SEQ ID NO.34 White matter, or
    With in sequence tableShown base sequence orThe egg of shown amino acid sequence White matter, or
    With in sequence tableShown base sequence orThe egg of shown amino acid sequence White matter, or
    Egg with the base sequence in sequence table shown in SEQ ID NO.17 or the amino acid sequence shown in SEQ ID NO.37 White matter, or
    Egg with the base sequence in sequence table shown in SEQ ID NO.18 or the amino acid sequence shown in SEQ ID NO.38 White matter, or
    Egg with the base sequence in sequence table shown in SEQ ID N0.19 or the amino acid sequence shown in SEQ ID NO.39 White matter, or
    Egg with the base sequence in sequence table shown in SEQ ID NO.20 or the amino acid sequence shown in SEQ ID NO.40 White matter, or
    Egg with the base sequence in sequence table shown in SEQ ID NO.21 or the amino acid sequence shown in SEQ ID NO.41 White matter, or
    Egg with the base sequence in sequence table shown in SEQ ID NO.22 or the amino acid sequence shown in SEQ ID NO.42 White matter, or
    Egg with the base sequence in sequence table shown in SEQ ID NO.23 or the amino acid sequence shown in SEQ ID NO.43 White matter, or
    Egg with the base sequence in sequence table shown in SEQ ID NO.24 or the amino acid sequence shown in SEQ ID NO.44 White matter, or
    With in sequence tableShown base sequence orThe egg of shown amino acid sequence White matter, or
    With in sequence tableShown base sequence orThe egg of shown amino acid sequence White matter, or
    Egg with the base sequence in sequence table shown in SEQ ID NO.27 or the amino acid sequence shown in SEQ ID NO.47 White matter.
CN201710395727.8A 2016-11-23 2017-05-27 Preparation method of high-yield shikimic acid mutant Active CN107446870B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102304539A (en) * 2011-08-26 2012-01-04 河南孟成生物药业股份有限公司 Construction method of genetic engineering strain for producing shikimic acid
CN102994439A (en) * 2012-12-12 2013-03-27 江南大学 Escherichia coli recombinant strain producing shikimic acid, and construction method and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102304539A (en) * 2011-08-26 2012-01-04 河南孟成生物药业股份有限公司 Construction method of genetic engineering strain for producing shikimic acid
CN102994439A (en) * 2012-12-12 2013-03-27 江南大学 Escherichia coli recombinant strain producing shikimic acid, and construction method and application thereof

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Title
BASHIR SAJO MIENDA等: "Model-guided metabolic gene knockout of gnd for enhanced succinate production in Escherichia coli from glucose and glycerol substrates", 《COMPUT BIOL CHEM》 *
LOPIAN L等: "Spatial and temporal organization of the E. coli PTS components", 《EMBO J》 *
李明明等: "理性设计和构建过量合成莽草酸的大肠杆菌代谢工程菌", 《生物工程学报》 *

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