CN105385702A - Acid-resistant threonine production bacterium and establishment method and application thereof - Google Patents
Acid-resistant threonine production bacterium and establishment method and application thereof Download PDFInfo
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- CN105385702A CN105385702A CN201510579005.9A CN201510579005A CN105385702A CN 105385702 A CN105385702 A CN 105385702A CN 201510579005 A CN201510579005 A CN 201510579005A CN 105385702 A CN105385702 A CN 105385702A
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Abstract
The invention provides an establishment method of an acid-resistant threonine production bacterium. The acid-resistant threonine production bacterium obtained through the method can be used for preparing threonine, the deamination reaction is conducted on glutamic acid through a glutamic acid oxidase gene, glutamic acid is converted into alpha-ketoglutarate, and generated free NH3 and H2O2 can neutralize redundant intracellular protons so that the acid-resistant capacity of thalli can be improved. The acid-resistant system can be effectively applied to fermentation production of biologics, the system can not generate gamma-aminobutyric acid or agmatine which has a thallus restraining effect, a gene for expressing gamma-aminobutyric acid or agmatine transport protein is not needed, and the burdens of recombined strains are reduced; alpha-oxoglutarate generated in the reaction can directly enter TCA circulation, energy is provided for thallus growth, the use amount of liquid ammonia is effectively decreased, and the influences of pH fluctuation on fermentation are effectively avoided.
Description
Technical field
The present invention relates to biological technical field, especially a kind of acid resistance Threonine produces bacterium and construction process thereof and application.
Background technology
Current intestinal bacteria have three kinds of known resistance to acid systems (ARs).AR
1the mechanism of system (generally also referred to as oxidation system) is not still understood, but what was certain was that it needs the gene of activated code σ SandCRP (receptor protein of cAMP), and suppressed by glucose.AR
2l-glutamic acid (Glu)/γ-aminobutyric acid (GABA) translocator GadC and two L-Glutamic decarboxylase GadA and GadB is comprised in system.Amino acid transporter GadC can change the outer Glu of born of the same parents and enter in cell, and form GABA under tying with the proton in born of the same parents the effect being incorporated in L-Glutamic decarboxylase, GABA is transported to outside born of the same parents under the effect of GadC again.Similar AR
2system, AR
3system is also made up of two portions, L-arginine (Arg)/agmatine (Agmatine) translocator AdiC and arginine decarboxylase AdiA.Arginine enters in born of the same parents by AdiC, with proton tie be incorporated in AdiA effect under form Agmatine, Agmatine is transported to outside born of the same parents by AdiC again.ARs system is activated in acid condition, increases pH value, improve the viability of intestinal bacteria at sour environment by transfer born of the same parents inner proton.
Under the inspiration of ARs system, the researchist of Tsing-Hua University constructs one
novelintestinal bacteria acid resistance system, by express amino acid reverse transport protein GadC, glutamine is converted into glutaminate and discharges free NH simultaneously by the L-Glutamine deaminase YbaS of acid activatable
3, then in conjunction with AR
2system improves colibacillary acid-fast ability.This YbaS and GadC can be activated by acid pH, and only suitably could play function when below pH6.0.
But existing report is just studied the acidproof mechanism of intestinal bacteria, is not applied in the fermentative production of biological product.Pass through AR simultaneously
2or AR
3system can produce the inhibited γ-aminobutyric acid of cell or agmatine, and γ-aminobutyric acid or agmatine are discharged to the separation and Extraction that born of the same parents also can have influence on object product outward.Therefore, AR is depended on
2or AR
3the acid resistance intestinal bacteria of system cannot really be applied in the production of industrially scalable.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of acid resistance Threonine to produce the construction process of bacterium.
Another technical problem to be solved by this invention is that the acid resistance Threonine providing the construction process producing bacterium by above-mentioned acid resistance Threonine to obtain produces bacterium.
Another technical problem to be solved by this invention is to provide above-mentioned acid resistance Threonine to produce the application of bacterium, for the preparation of Threonine.
For solving the problems of the technologies described above, technical scheme of the present invention is:
Acid resistance Threonine produces the construction process of bacterium, with Threonine produce bacterium E.coliTHRD (
preservationnumber CGMCCNo.11074) be research object, by molecular modification, process LAN synthesis L-GLOD gene on pTrc99A plasmid, or integrate synthesis L-GLOD gene to E.coliTHRD genome, obtain acid resistance threonine producing strain.
Preferably, above-mentioned acid resistance Threonine produces the construction process of bacterium, and concrete steps are as follows:
(1) L-GLOD (LGOX) gene fragment is synthesized;
(2) be connected after L-GLOD (LGOX) gene and expression vector pTrc99A thereof being carried out double digestion with Hind III with BamH I, connect product conversion in E.coliTHRD; Or
Utilize Red homologous recombination gene knockout method, integrate LGOX gene on the pectinose gene araABD site of E.coliTHRD;
Wherein, the nucleotides sequence of described L-GLOD LGOX is classified as sequence shown in sequence table <400>1, the nucleotides sequence of described pectinose gene araABD is classified as sequence shown in sequence table <400>2
The genetic engineering bacterium that gained expresses L-GLOD is acid resistance Threonine production bacterium.
The acid resistance Threonine that the construction process producing bacterium by above-mentioned acid resistance Threonine obtains produces bacterium, is that process LAN L-GLOD gene or integration L-GLOD gene produce bacterial strain to the acid resistance in E.coliTHRD genome pectinose site on pTrc99A plasmid.
Above-mentioned acid resistance Threonine is produced bacterium and is being prepared the application in Threonine.
Preferably, above-mentioned acid resistance Threonine produces the application of bacterium, and the concrete steps preparing Threonine are as follows:
(1) shake flask fermentation: 1. seed culture: described acid resistance Threonine is produced bacterium and be inoculated in 9 layers of gauze sealing in the 500mL round bottom triangular flask that 30-50mL seed culture medium is housed from picking activated inclined plane 2 ring, be placed on rotary shaker, 32-37 DEG C, 100-250rpm shaking culture 6-10h; 2. shake flask fermentation: by 5-10% inoculum size by step 1. in inoculum access be equipped with in the 500mL baffle flask of 30-50mL fermention medium; With purpurum bromocresolis be indicator at pH5.2, be that indicator carries out shake flask fermentation, leavening temperature 32-37 DEG C, rotating speed 100-250rpm, fermentation period 26-30h under pH6.0 condition with dibromothymolsulfonphthalein;
(2) ferment tank: 1. seed culture: 8-10mL sterilized water is joined in 3 activated inclined planes and prepare bacteria suspension, all bacteria suspension accesses are equipped with in the 5L fermentor tank of 1.5L seed culture medium, the ammoniacal liquor that stream adds 25% (W/V) regulates fermented liquid pH to 6.8-7.2, dissolved oxygen maintains 30-50%, ventilation 3-5m
3/ h, cultivates 6-8h for mixing speed 200-600rpm, 35-37 DEG C; 2. ferment tank is cultivated: with 10%-13% inoculum size by step 1. in inoculum be connected to the 5L fermentor tank that 3L fermention medium is housed and carry out fermentation culture, leavening temperature 35-37 DEG C, ventilation 3-5m
3/ h, mixing speed 300-1000rpm, dissolved oxygen maintains 30-60%, and stream adds the glucose solution that concentration is 60-80% (W/V), and maintenance remaining sugar concentration is 0.1-0.5% (W/V); The ammoniacal liquor that earlier fermentation stream adds 25% (W/V) controls fermented liquid pH to 6.8-7.2, and the ammoniacal liquor that fermentation middle and later periods stream adds 25% (W/V) controls fermented liquid pH to 5.8-6.2, fermentation period 26-28h.
Preferably, above-mentioned acid resistance Threonine produces the application of bacterium, and the substratum that described activated inclined plane uses is: glucose 0.5-1g/L, peptone 5-10g/L, extractum carnis 2.5-5g/L, yeast powder 2.5-5g/L, NaCl1-2.5g/L, agar strip 10-20g/L.
Preferably, above-mentioned acid resistance Threonine produces the application of bacterium, and in described step (1), (2), seed culture based component is: glucose 15-25g/L, yeast powder 5-10g/L, Tryptones 3-6g/L, (NH4)
2sO
41-2g/L, KH
2pO
40.6-1.2g/L, MgSO47H
2o0.3-0.5g/L, FeSO
47H
2o5-10mg/L, MnSO
4h
2o5-10mg/L, vitamin B group (VB
1, VB
3, VB
5, VB
7, VB
12) 0.5-1mg/L, VH0.15-0.3mg/L.
Preferably, above-mentioned acid resistance Threonine produces the application of bacterium, and in described step (1), (2), fermentation medium components is: glucose 25-40g/L, yeast powder 1-2g/L, Tryptones 2-4g/L, Trisodium Citrate 0.5-1g/L, L-glutamic acid 3-5g/L, KH
2pO
41-2g/L, MgSO
47H
2o0.4-0.7g/L, FeSO
47H
2o30-50mg/L, MnSO
4h
2o30-50mg/L, vitamin B group (VB
1, VB
3, VB
5, VB
7, VB
12) 0.6-0.8mg/L, VH0.1-0.2mg/L, in shaking flask, fermention medium dilute sulphuric acid regulates initial pH value to 5.2 or 6.0, fermentation cylinder for fermentation initial pH value of medium 7.0,115 DEG C of sterilizing 20min.
Preferably, above-mentioned acid resistance Threonine produces the application of bacterium, and in gained fermented liquid, the detection method of Threonine is as follows:
1. fermentation liquor pretreatment: get 1mL fermented liquid in 1.5mL centrifuge tube, the centrifugal 5min of 12000r/min, gets supernatant liquor and crosses 0.22 μm of filter membrane;
2. deriving method: got film secondary fermentation liquid 10 μ L in 2mL centrifuge tube, and added 200 μ L, the NaHCO of 4.2g/L
3solution, adds 200 μ L after mixing, the acetonitrile solution mixing of the DNF (DNFB) containing 1%, is placed in 60 DEG C of water bath dark place water bath with thermostatic control 1h, takes out after being cooled to room temperature, add the KH of 6.8g/L by centrifuge tube
2pO
4solution is settled to 1.3mL, mixing sample introduction;
3. high performance liquid chromatography detects: chromatographic column is AgilentZORBAXEclipseAAA, and moving phase is sodium acetate-acetonihile gradient elution, sees
table a, flow velocity 1.0mL/min, column temperature 33 DEG C, determined wavelength 360nm.
table agradient curve
The invention has the beneficial effects as follows:
Above-mentioned acid resistance Threonine is produced bacterium and is expressed L-GLOD gene LGOX by producing in bacterium E.coliTHRD at Threonine, and Pidolidone is oxidized to α-ketoglutaric acid, generates free NH simultaneously
3and H
2o
2; α-ketoglutaric acid can continue to participate in TCA and circulate as thalli growth provides energy, NH
3and H
2o
2then can in and unnecessary proton thus improve the acid-fast ability of thalline in born of the same parents; The acid resistance intestinal bacteria that above-mentioned transformation obtains can effectively be applied in the fermentative production of Threonine;
The present invention, by devising a kind of resistance to acid system of novelty for the preparation of Threonine, utilizes L-GLOD gene pairs L-glutamic acid to carry out deamination reaction, is translated into α-ketoglutaric acid, the free NH of generation
3and H
2o
2then can in and unnecessary proton thus improve the acid-fast ability of thalline in born of the same parents, described resistance to acid system can effectively be applied in the fermentative production of biological product.
Compared with traditional resistance to acid system, this system can not produce the inhibited γ-aminobutyric acid of thalline or agmatine, does not need the gene of expressing γ-aminobutyric acid or agmatine translocator, decreases the burden of recombinant bacterial strain; The α-ketoglutaric acid generated in reaction directly can enter TCA circulation, for thalli growth provides energy, therefore can not impact the separation and Extraction of object product; Utilize acid resistance Escherichia coli fermentation to produce Threonine, effectively can reduce the consumption of liquefied ammonia, and effectively prevent pH fluctuation to the impact caused of fermenting, be applicable to industrial applications.
Accompanying drawing explanation
fig. 1for the double digestion of plasmid pTrc99A-LGOX is verified
figure, wherein, M:Marker1:Hind III and BamH I double digestion pTrc99A-LGOX;
fig. 2for the integration of foreign gene LGOX
figure.
preservationinformation
Classification noun: colon bacillus Escherichiacoli
preservationorganization: Chinese microorganism strain
preservationmanagement
the councilcommon micro-organisms center
preservationunit address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
preservationdate: on July 14th, 2015
preservationnumber: CGMCCNo.11074
Embodiment
In order to make those skilled in the art better understand technical scheme of the present invention, below in conjunction with embodiment, technical scheme of the present invention is described in further detail.
Embodiment 1
The construction process of acid resistance threonine producing strain E.coliTHRD-pTrc99A-LGOX.
(1) L-GLOD LGOX gene fragment is synthesized;
(2) with the pET-His-LGOX plasmid synthesized for template, through restriction enzyme Hind III and BamH I respectively double digestion reclaim L-GLOD LGOX fragment; Employing T4 ligase enzyme is connected to the expression vector pTrc99A that same enzyme is cut, and adopts CaCl
2method is converted in E.coliDH5 α competence, coats on the LB solid medium containing 100ug/mL ammonia benzyl mycin, cultivates 12h for 37 DEG C; The switching of picking list bacterium colony, containing in the 15mL test tube of LB substratum, is extracted after 37 DEG C of cultivation 12h and is obtained recombinant plasmid pTrc99A-LGOX;
(3) CaCl is adopted
2method recombinant plasmid pTrc99A-LGOX is proceeded to Threonine produce bacterium E.coliTHRD (
preservationnumber CGMCCNo.11074) in competence, coat on the LB solid medium containing 100ug/mL ammonia benzyl mycin, cultivate 12h for 37 DEG C; The bacterium colony that picking grows carries out bacterium colony PCR checking, will be accredited as positive transformant access LB liquid nutrient medium, and extract plasmid and carry out digestion verification after 37 DEG C of cultivation 12h; The result
as Fig. 1shown in, show that recombinant plasmid pTrc99A-LGOX successfully proceeds in E.coliTHRD, obtain acid resistance Threonine and produce bacterium E.coliTHRD-pTrc99A-LGOX.
Embodiment 2
The construction process of acid resistance threonine producing strain E.coliTHRDaraABD::LGOX.
(1) L-GLOD LGOX gene fragment is synthesized;
(2) adopt round pcr using THRD genome for template amplification Arabinose promoter pBAD is as upstream homology arm, and amplification pectinose gene sites downstream is as downstream homology arm;
(3) the chlorampenicol resistant fragment on round pcr amplification pKD3 is adopted;
(4) according to gene order design homology arm primer, the amplified fragments obtained with step (1) (2) (3) is that template obtains integration fragment by over-lap PCR, described integration fragment is made up of pBAD upstream homology arm, LGOX gene fragment, chloramphenicol resistance gene fragment, downstream homology arm
as Fig. 2shown in;
(5) obtaining positive transformant by integrating in the electric E.coliTHRD be converted into containing pKD46 plasmid of fragment, after eliminating the chloramphenicol resistance gene in transformant, obtaining the acid resistance Threonine production bacterium E.coliTHRDaraABD::LGOX that pectinose gene araABD replaces with LGOX gene.
The nucleotides sequence of Arabinose promoter pBAD is classified as sequence shown in sequence table <400>3.
The nucleotides sequence of chloramphenicol resistance gene fragment is classified as sequence shown in sequence table <400>4, and the nucleotides sequence of homology arm segments downstream is classified as sequence shown in sequence table <400>5.
Embodiment 3
The shake flask fermentation of acid resistance threonine producing strain E.coliTHRD-pTrc99A-LGOX is cultivated and is detected
(1) seed culture: E.coliTHRD-pTrc99A-LGOX and E.coliTHRD is inoculated in 9 layers of gauze sealing in the 500mL round bottom triangular flask that 30mL seed culture medium is housed from picking activated inclined plane 2 ring, be placed on rotary shaker, 36 DEG C, 200rpm shaking culture 8h;
The substratum that activated inclined plane uses is: glucose 1g/L, peptone 10g/L, extractum carnis 5g/L, yeast powder 5g/L, NaCl2.5g/L, agar strip 20g/L;
Seed culture based component is: glucose 25g/L, yeast powder 10g/L, Tryptones 6g/L, (NH4) 2SO42g/L, KH2PO41.2g/L, MgSO47H2O0.5g/L, FeSO47H2O10mg/L, MnSO4H2O10mg/L, vitamin B group (VB1, VB3, VB5, VB7, VB12) 1mg/L, VH0.3mg/L;
(2) shake flask fermentation: the inoculum access in step (1) is equipped with in the 500mL baffle flask of 30mL fermention medium by 10% inoculum size.With purpurum bromocresolis be indicator at pH5.2, be that indicator carries out shake flask fermentation under pH6.0 condition with dibromothymolsulfonphthalein, leavening temperature 37 DEG C, rotating speed 200rpm, fermentation period 28h.
Fermentation medium components is: glucose 40g/L, yeast powder 2g/L, Tryptones 4g/L, Trisodium Citrate 1g/L, L-glutamic acid 5g/L, KH2PO42g/L, MgSO47H2O0.7g/L, FeSO47H2O50mg/L, MnSO4H2O50mg/L, vitamin B group (VB1, VB3, VB5, VB7, VB12) 0.8mg/L, VH0.2mg/L, regulate initial pH value to 5.2 or 6.0 with dilute sulphuric acid.
(3) fermentation results detects: 1. fermentation liquor pretreatment: get 1mL fermented liquid in 1.5mL centrifuge tube, the centrifugal 5min of 12000r/min, gets supernatant liquor and cross 0.22 μm of filter membrane.2. deriving method: got film secondary fermentation liquid 10 μ L in 2mL centrifuge tube, and added 200 μ L, the NaHCO of 4.2g/L
3solution, adds 200 μ L after mixing, the acetonitrile solution mixing of the DNF (DNFB) containing 1%, is placed in 60 DEG C of water bath dark place water bath with thermostatic control 1h, takes out after being cooled to room temperature, add the KH of 6.8g/L by centrifuge tube
2pO
4solution is settled to 1.3mL, mixing sample introduction.3. high performance liquid chromatography detects: chromatographic column is AgilentZORBAXEclipseAAA, and moving phase is sodium acetate-acetonihile gradient elution, sees
table 1, flow velocity 1.0mL/min, column temperature 33 DEG C, determined wavelength 360nm.Detected result is shown in
table 2.
Shake flat experiment result proves that being imported with of L-GLOD gene helps improve the throughput of fermentation strain under condition of acidic pH.Under pH5.2 condition, E.coliTHRD-pTrc99A-LGOX dry cell weight and production amount of threonine are respectively 1.58 and 1.64 times of former bacterium; Under pH6.0 condition, E.coliTHRD-pTrc99A-LGOX dry cell weight and production amount of threonine are respectively 1.65 and 1.67 times of former bacterium.
table 1gradient curve
table 2shake flask fermentation result
Embodiment 4
The upper tank fermentation culture of acid resistance threonine producing strain E.coliTHRD-pTrc99A-LGOX and E.coliTHRDaraABD::LGOX and detection
(1) seed culture: 10mL sterilized water is joined in 3 activated inclined planes and prepare bacteria suspension, all bacteria suspension accesses are equipped with in the 5L fermentor tank of 1.5L seed culture medium, the ammoniacal liquor that stream adds 25% (W/V) regulates fermented liquid pH to 7.0, and dissolved oxygen maintains 30%, ventilation 4m
3/ h, mixing speed 400rpm, cultivate 8h for 36 DEG C.
Activated inclined plane substratum and seed culture medium are with embodiment 3.
(2) ferment tank is cultivated: with 11% inoculum size, the inoculum in step (1) is connected to the 5L fermentor tank that 3L fermention medium is housed and carries out fermentation culture, leavening temperature 37 DEG C, ventilation 5m
3/ h, mixing speed 600rpm, dissolved oxygen maintains 40%, and stream adds the glucose solution that concentration is 70% (W/V), and maintaining remaining sugar concentration is 0.3% (W/V).The ammoniacal liquor that earlier fermentation stream adds 25% (W/V) controls fermented liquid pH to 7.0, and the ammoniacal liquor that fermentation middle and later periods stream adds 25% (W/V) controls fermented liquid pH to 6.0, fermentation period 28h.
Fermentation medium components is: glucose 40g/L, yeast powder 2g/L, Tryptones 4g/L, Trisodium Citrate 1g/L, L-glutamic acid 5g/L, KH2PO42g/L, MgSO47H2O0.7g/L, FeSO47H2O50mg/L, MnSO4H2O50mg/L, vitamin B group (VB1, VB3, VB5, VB7, VB12) 0.8mg/L, VH0.2mg/L.
(3) fermentation results detects: detection method is with embodiment 3.Detected result is shown in
table 3.
table 3upper tank fermentation results
Upper tank result shows, expressing the genetic engineering bacterium of L-GLOD can more effective accumulation Threonine under the condition of pH6.0, the dry cell weight of genetic engineering bacterium and former bacterium is basically identical, but E.coliTHRD-pTrc99A-LGOXL-production amount of threonine and the more former bacterium of thalline glucose acid invert ratio improve 10.00% and 10.91% respectively; E.coliTHRDaraABD::LGOXL-production amount of threonine and the more former bacterium of thalline glucose acid invert ratio improve respectively and improve 6.00% and 5.60% respectively.
Above-mentionedly with reference to embodiment, the detailed description that bacterium and construction process and application thereof carry out is produced to this kind of Threonine; illustrative instead of determinate; several embodiments can be listed according to institute's limited range; therefore in the change do not departed under general plotting of the present invention and amendment, should belong within protection scope of the present invention.
Claims (8)
1. the construction process of an acid resistance Threonine production bacterium, it is characterized in that: produce bacterium E.coliTHRD (preserving number CGMCCNo.11074) for research object with Threonine, pass through molecular modification, process LAN L-GLOD gene on pTrc99A plasmid, or integrate L-GLOD gene to E.coliTHRD genome, obtain acid resistance threonine producing strain.
2. acid resistance Threonine according to claim 1 produces the construction process of bacterium, it is characterized in that: concrete steps are as follows:
(1) L-GLOD gene fragment is synthesized;
(2) be connected after L-GLOD gene and expression vector pTrc99A thereof being carried out double digestion with Hind III with BamH I, connect product conversion in E.coliTHRD; Or
Utilize Red homologous recombination gene knockout method, integrate L-GLOD gene on the pectinose gene araABD site of E.coliTHRD;
Wherein, the nucleotides sequence of described L-GLOD is classified as sequence shown in sequence table <400>1, the nucleotides sequence of described pectinose gene araABD is classified as sequence shown in sequence table <400>2
The genetic engineering bacterium that gained expresses L-GLOD is acid resistance Threonine production bacterium.
3. the acid resistance Threonine that the construction process producing bacterium by acid resistance Threonine described in claim 1 or 2 obtains produces bacterium, is that process LAN L-GLOD gene or integration L-GLOD gene produce bacterial strain to the acid resistance in E.coliTHRD genome pectinose site on pTrc99A plasmid.
4. described in claim 3, acid resistance Threonine production bacterium is preparing the application in Threonine.
5. acid resistance Threonine produces the application of bacterium according to claim 4, it is characterized in that: the concrete steps preparing Threonine are as follows:
(1) shake flask fermentation: 1. seed culture: described acid resistance Threonine is produced bacterium and be inoculated in 9 layers of gauze sealing in the 500mL round bottom triangular flask that 30-50mL seed culture medium is housed from picking activated inclined plane 2 ring, be placed on rotary shaker, 32-37 DEG C, 100-250rpm shaking culture 6-10h; 2. shake flask fermentation: by 5-10% inoculum size by step 1. in inoculum access be equipped with in the 500mL baffle flask of 30-50mL fermention medium; With purpurum bromocresolis be indicator at pH5.2, be that indicator carries out shake flask fermentation, leavening temperature 32-37 DEG C, rotating speed 100-250rpm, fermentation period 26-30h under pH6.0 condition with dibromothymolsulfonphthalein;
(2) ferment tank: 1. seed culture: 8-10mL sterilized water is joined in 3 activated inclined planes and prepare bacteria suspension, all bacteria suspension accesses are equipped with in the 5L fermentor tank of 1.5L seed culture medium, the ammoniacal liquor that stream adds 25% (W/V) regulates fermented liquid pH to 6.8-7.2, dissolved oxygen maintains 30-50%, ventilation 3-5m
3/ h, cultivates 6-8h for mixing speed 200-600rpm, 35-37 DEG C; 2. ferment tank is cultivated: with 10%-13% inoculum size by step 1. in inoculum be connected to the 5L fermentor tank that 3L fermention medium is housed and carry out fermentation culture, leavening temperature 35-37 DEG C, ventilation 3-5m
3/ h, mixing speed 300-1000rpm, dissolved oxygen maintains 30-60%, and stream adds the glucose solution that concentration is 60-80% (W/V), and maintenance remaining sugar concentration is 0.1-0.5% (W/V); The ammoniacal liquor that earlier fermentation stream adds 25% (W/V) controls fermented liquid pH to 6.8-7.2, and the ammoniacal liquor that fermentation middle and later periods stream adds 25% (W/V) controls fermented liquid pH to 5.8-6.2, fermentation period 26-28h.
6. acid resistance Threonine according to claim 5 produces the application of bacterium, it is characterized in that: the substratum that described activated inclined plane uses is: glucose 0.5-1g/L, peptone 5-10g/L, extractum carnis 2.5-5g/L, yeast powder 2.5-5g/L, NaCl1-2.5g/L, agar strip 10-20g/L.
7. acid resistance Threonine according to claim 5 produces the application of bacterium, it is characterized in that: in described step (1), (2), seed culture based component is: glucose 15-25g/L, yeast powder 5-10g/L, Tryptones 3-6g/L, (NH4)
2sO
41-2g/L, KH
2pO
40.6-1.2g/L, MgSO47H
2o0.3-0.5g/L, FeSO
47H
2o5-10mg/L, MnSO
4h
2o5-10mg/L, vitamin B group (VB
1, VB
3, VB
5, VB
7, VB
12) 0.5-1mg/L, VH0.15-0.3mg/L.
8. acid resistance Threonine according to claim 5 produces the application of bacterium, it is characterized in that: in described step (1), (2), fermentation medium components is: glucose 25-40g/L, yeast powder 1-2g/L, Tryptones 2-4g/L, Trisodium Citrate 0.5-1g/L, L-glutamic acid 3-5g/L, KH
2pO
41-2g/L, MgSO
47H
2o0.4-0.7g/L, FeSO
47H
2o30-50mg/L, MnSO
4h
2o30-50mg/L, vitamin B group (VB
1, VB
3, VB
5, VB
7, VB
12) 0.6-0.8mg/L, VH0.1-0.2mg/L, in shaking flask, fermention medium dilute sulphuric acid regulates initial pH value to 5.2 or 6.0, fermentation cylinder for fermentation initial pH value of medium 7.0,115 DEG C of sterilizing 20min.
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| CN106399343A (en) * | 2015-07-29 | 2017-02-15 | 上海交通大学 | Biological improvement synthesis method of glutaric acid |
| CN106867952A (en) * | 2017-01-09 | 2017-06-20 | 天津科技大学 | One plant of Recombinant organism and the method using its production L threonine |
| CN110229774A (en) * | 2019-07-02 | 2019-09-13 | 山东国力生物科技有限公司 | A kind of arginine decarboxylase gene engineering bacteria and its high density fermentation culture method |
| CN110846351A (en) * | 2019-12-22 | 2020-02-28 | 赵兰坤 | Threonine fermentation medium prepared by using mycoprotein as raw material |
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| CN101580813A (en) * | 2008-05-12 | 2009-11-18 | 长春大成实业集团有限公司 | Method for producing L-threonine by fermentation |
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Patent Citations (1)
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| CN101580813A (en) * | 2008-05-12 | 2009-11-18 | 长春大成实业集团有限公司 | Method for producing L-threonine by fermentation |
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| JIN-HO LEE等: "Global Analyses of Transcriptomes and Proteomes of a Parent Strain and an L-Threonine-Overproducing Mutant Strain", 《JOURNAL OF BACTERIOLOGY》 * |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399343A (en) * | 2015-07-29 | 2017-02-15 | 上海交通大学 | Biological improvement synthesis method of glutaric acid |
| CN106399343B (en) * | 2015-07-29 | 2019-07-05 | 上海交通大学 | Glutaric acid biology improves synthetic method |
| CN106867952A (en) * | 2017-01-09 | 2017-06-20 | 天津科技大学 | One plant of Recombinant organism and the method using its production L threonine |
| CN106867952B (en) * | 2017-01-09 | 2019-10-18 | 天津科技大学 | One plant of Recombinant organism and the method for producing L-threonine using it |
| CN110229774A (en) * | 2019-07-02 | 2019-09-13 | 山东国力生物科技有限公司 | A kind of arginine decarboxylase gene engineering bacteria and its high density fermentation culture method |
| CN110229774B (en) * | 2019-07-02 | 2020-12-08 | 山东国力生物科技有限公司 | Arginine decarboxylase gene engineering bacterium and high-density fermentation culture method thereof |
| CN110846351A (en) * | 2019-12-22 | 2020-02-28 | 赵兰坤 | Threonine fermentation medium prepared by using mycoprotein as raw material |
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| CN105385702B (en) | 2019-06-14 |
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Address after: No.9, 13th Street, economic and Technological Development Zone, Binhai New Area, Tianjin Patentee after: Tianjin University of Science and Technology Address before: No.1038 Dagu South Road, Hexi District, Tianjin Patentee before: Tianjin University of Science and Technology |
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