CN105671008A - Pyrimidine nucleoside high-yielding strain and carbamyl phosphate synthetase adjusting site thereof - Google Patents
Pyrimidine nucleoside high-yielding strain and carbamyl phosphate synthetase adjusting site thereof Download PDFInfo
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- C12P19/00—Preparation of compounds containing saccharide radicals
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- C12Y603/05—Carbon-nitrogen ligases with glutamine as amido-N-donor (6.3.5)
- C12Y603/05005—Carbamoyl-phosphate synthase (glutamine-hydrolysing) (6.3.5.5)
Abstract
The invention belongs to the technical field of enzyme engineering, and concretely relates to a pyrimidine nucleoside high-yielding strain and a carbamyl phosphate synthetase adjusting site thereof. The invention provides a pyrimidine nucleoside production Bacillus subtilis mutant strain and a carbamyl phosphate synthetase encoding gene. A key regulation site related to carbamyl phosphate synthetase end product feedback inhibition is known, and provides reference for breeding of later pyrimidine nucleoside high-yielding strains. The Bacillus subtilis mutant strain allows the output of fermentation process produced nucleoside pyrimidine uridine to reach 12.5-14.5g/L, and has substantially improved tolerance to different pyrimidine nucleoside structure analogs.
Description
Technical field:
The invention belongs to technical field of enzyme engineering, it is specifically related to bacterial strain and the carbamyl phosphate synthetase regulatory site thereof of a strain high yield pyrimidine nucleoside.
Background technology:
Pyrimidine nucleoside comprises urine glycosides and cytidine, is the important composition composition of all living things body, and their purposes are extensive, especially as the intermediate of antiviral antitumor drug, occupy critical role at field of medicaments. The production method of pyrimidine nucleoside has chemical synthesis, hydrolysis RNA method and microbe fermentation method, the relative merits of comprehensive several method, microbe fermentation method because its cycle is short, easy to control, product rate height, pollute the advantages such as little, the potentiality with heavy industrialization, are therefore subject to people's attention day by day.
The seed selection that pyrimidine nucleoside produces bacterial strain starts from the sixties in 20th century, and the seed selection of relevant pyrimidine nucleoside superior strain both at home and abroad is all the method for mutagenesis integrated structure analogue resistant strain screening substantially, and wherein research with the chemical company in military field of Japan is the most outstanding.
Wu Tian company is first in the process of high yield urine glycosides strain improvement is starting strain taking wild-type B. subtilis, through NTG mutagenesis, select a strain uracil-deficient type bacterial strain No.122, this bacterial strain is providing in 50mg/L uridylic situation, it is possible to accumulated whey acid 8.58g/L (50mM) vitamin B13 and 7.78g/L (27mM) orotidine; On the basis of No.122, again through NTG mutagenesis, the substratum containing 300mg/L6-azauracil screens bacterial strain No.258, this strain for accumulating urine glycosides 10g/L, uridylic 7g/L. Continue NTG mutagenesis, in the 6-azauracil substratum containing 5g/L, screen bacterial strain No.508, this strain for accumulating urine glycosides 46g/L, uridylic 6g/L. Finally, in order to reduce the accumulation volume of by product uridylic, proceed mutagenesis, screen the bacterial strain No.556 of a strain Uridine phosphorylase almost loss of activity, urine glycosides 55g/L can be accumulated, uridylic accumulation volume < 1g/L, by the optimization to culture condition and medium component, stable accumulation urine glycosides 65g/L in 6000L fermentor tank.
They to the seed selection of high yield cytidine bacterial classification equally based on No.122 bacterial strain. First by, after NTG mutagenesis, filtering out cytidine deaminase deletion mycopremna No.229 one by one, accumulation cytidine 0.2g/L, urine glycosides 1g/L, namely this bacterial strain loses energy for growth under the existence of 50mg/L5-fluorine cytidine.Therefore, after continuing mutagenesis, obtain the bacterial strain No.344 that a strain can tolerate 500mg/L5-fluorine cytidine, this strain for accumulating cytidine 10.4g/L. Learning through enzymic activity confirmatory experiment, this bacterial strain is compared with No.122 bacterial strain, and the activity of carbamyl phosphate synthetase and CTP synthetic enzyme all improves more than ten times. Through the further mutagenic treatment to this bacterial strain, filter out and can tolerate 12g/L3-removing impurities azauracil bacterial strain No.428, it is possible to accumulation cytidine 14.2g/L, this bacterium relieves the feedback regulation effect suffered by CTP synthetic enzyme substantially. Afterwards, in No.428, introduce sudden change by homologous recombination, obtain the carbamyl phosphate synthetase bacterial strain No.515 not being subject to feedback inhibition, make cytidine output reach 18.8g/L. Again through NTG mutagenesis, obtaining a strain homoserine dehydrogenase deletion mycopremna No.615, this bacterial strain can accumulate cytidine 23.5g/L, finally by the adjusting and optimizing to carbon source glucose concentration, this bacterial strain can accumulate cytidine 30.2g/L in 60 tons of fermentor tanks, and stable yield.
And although Wu Tian company of Japan achieves great achievement at the selection of pyrimidine nucleoside superior strain, the bacterial classification of its seed selection produces glycosides ability and also holds a safe lead in the world, but limiting by technical qualification at that time, they do not point out the crucial regulatory site of the carbamyl phosphate synthetase closely bound up with pyrimidine nucleoside high yield.
B.subtilis has two kinds of carbamyl phosphate synthetases (EC6.3.5.5, carbamoylphosphatesynthetase, CPSase): one is pyrAA/pyrAB genes encoding, the first step reaction of catalysis UMP de novo synthesis approach; Two is carA/carB genes encoding, participates in Arginine biosynthesis. Although, these two kinds of carbamyl phosphate synthetases all catalysis glutamine and CO2Reaction, consumes 2 molecule ATP and generates carbamyl phosphate. But, two carbamyl phosphate synthetase physiological functions of B.subtilis are different, and their expression and enzymic activity regulation mechanism are also completely different.
PyrAA/pyrAB be positioned at pyr operon the 5th and 6, coding synthesizes relevant carbamyl phosphate synthetase with UMP. Wherein pyrAA mrna length is 1095bp, and the little sub-base of encoding carbamoyl phosphate synthetase, the epsilon-amino of catalysis glutamine transfers to Dare's base. PyrAB mrna length is 3216bp, Dare's base of encoding carbamoyl phosphate synthetase, catalysis MgATP, CO2With NH3Synthesis carbamyl phosphate. PyrAA/pyrAB belongs to pyr operon, and its expression also activates by PRPP, by the feedback repression of UMP, UDP and UTP. The carbamyl phosphate synthetase of pyrAA/pyrAB coding or an allosteric enzyme, its enzyme is lived by UMP feedback inhibition, activates by PRPP, and needs Mg2+Existence.
The difficult point of pyrimidine nucleoside superior strain seed selection is to release feedback repression and the feedback inhibition effect of end product, the particularly releasing of feedback inhibition effect, although researchist has disclosed the concrete regulatory mechanism of pyrimidine nucleoside anabolism, and it is not from molecular level, set forth the mechanism that pyrimidine operon checks by end product, but but clear for the regulatory site that the carbamyl phosphate synthetase by end product feedback inhibition is concrete.
Along with the application of molecular biology in Microbial Breeding, also the method attempting utilizing PCR method to introduce point mutation about researchist releases the feedback inhibition suffered by carbamyl phosphate synthetase, but owing to the research of carbamyl phosphate synthetase structure and function is not too deep, the method that PCR introduces point mutation is restricted in the application released in feedback inhibition suffered by carbamyl phosphate synthetase, it has been reported that site be not too obvious to the releasing effect of feedback inhibition effect.
The present invention provides a strain by the mutagenic and breeding subtilis with industrial applications potentiality out, it is possible to for the correlative study of fermentative Production pyrimidine nucleoside; To the order-checking of the genes involved of bacterial strain and after sequence alignment, specify that and carbamyl phosphate synthetase by the relevant crucial regulatory site of end product feedback inhibition, it is possible to be the seed selection offer reference of later pyrimidine nucleoside superior strain.
Summary of the invention:
One of technical scheme that the present invention solves the problem: be to provide a bacillus subtilis mutant strain, the 1016th amino acids of described mutant strain carbamyl phosphate synthetase Dare base is leucine.
Described bacillus subtilis mutant strain is specially subtilis (Bacillussubtilis) A219. This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 2nd, 2015, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, postcode 100101, deposit number is CGMCCNo.11774.
Described subtilis A219 is taking the subtilis of a strain accumulation pyrimidine nucleoside as starting strain, selected by atmospheric pressure at room plasma mutagenesis and pyrimidine nucleoside analog resistant strain, concrete steps are as follows: taking produce pyrimidine nucleoside withered grass gemma TD131 as starting strain, carry out atmospheric pressure at room plasma mutagenesis, then bacterial strain T823 is being filtered out containing on 300mg/L2-thiouracil minimum medium, then it is starting strain taking T823, after atmospheric pressure at room plasma mutagenesis, strains A 219 is being filtered out containing on 100mg/L6-azauracil minimum medium.
The pyrimidine nucleoside operon of mutant strain A219 is checked order, and carry out alignment with starting strain TD131, find that the encoding gene of the carbamyl phosphate synthetase of mutant strain A219 is undergone mutation, mutational site is at Dare's base (sub-base in regulatory site place, by pyrAB genes encoding) on, in A219, pyrAB gene the 3047th bit base is undergone mutation, cytosine(Cyt) turns into thymus pyrimidine, causing the proline(Pro) of 1016, carbamyl phosphate synthetase Dare base to be changed into leucine, other gene of pyrimidine nucleoside operon does not find sudden change.
The nucleotide sequence of original pyrAB gene is as shown in sequence table SEQ IDNo.1;
Original carbamyl phosphate synthetase Dare base aminoacid sequence is as shown in sequence table SEQ IDNo.3;
The nucleotide sequence of sudden change pyrAB gene is as shown in sequence table SEQ IDNo.2;
Carbamyl phosphate synthetase Dare base variant amino acid sequence is as shown in sequence table SEQ IDNo.4;
Nucleotide sequence described in the corresponding sequence table SEQ IDNo.1 of the Position Number of nucleotide of the present invention;
Aminoacid sequence described in the corresponding sequence table SEQ IDNo.3 of amino acid whose Position Number in the present invention.
The two of the technical scheme that the present invention solves the problem: the big subunit coding gene being to provide a kind of carbamyl phosphate synthetase, the nucleotide sequence of described gene is as shown in SEQIDNo.2.
The three of the technical scheme that the present invention solves the problem: the fermentation method for producing being to provide a kind of pyrimidine nucleoside, it is specially: be produce bacterial strain taking A219, inoculum size is 10%, and culture temperature is 34-36 DEG C, pH6.7-7.0, molten oxygen controls at 20-30%, residual sugar controls at 0.5-1%, and fermentation period 48h, after having fermented, fermented liquid is urinated glycosides concentration and can reach 13.5 ± 1g/L, be 3.3 times of starting strain TD131 urine glycosides output (4.1 ± 0.5g/L).
Fermention medium (g/L): glucose 50, yeast powder 5, corn steep liquor 20, SODIUMNITRATE 15, potassium primary phosphate 1, dipotassium hydrogen phosphate 5.2, magnesium sulfate 5, ammonium sulfate 5, Trisodium Citrate 10, Sodium Glutamate 10, calcium chloride 1, manganous sulfate 0.02, zinc sulfate 0.02, pH6.7～7.0, the independent sterilizing of corn steep liquor, the independent sterilizing of calcium salt, magnesium manganese zinc salt is made into mixing solutions sterilizing together, glucose is made into the 80% independent sterilizing of solution, the sterilizing together of all the other components, sterilising conditions corn steep liquor is 0.1MPa, 20min, all the other are 0.075MPa, 15min.
Useful effect:
1, the present invention provides a strain and produces the bacillus subtilis mutant strain of pyrimidine nucleoside and the encoding gene of a carbamyl phosphate synthetase, specify that a crucial regulatory site being subject to end product feedback inhibition relevant to carbamyl phosphate synthetase, it is possible to be the seed selection offer reference of later pyrimidine nucleoside superior strain.
2, the present invention provides the bacillus subtilis mutant strain that pyrimidine nucleoside is produced in a strain, urinates glycosides output by fermentative Production nucleoside pyrimidine and can reach 13.5 ± 1g/L, is 3.3 times of starting strain TD131 urine glycosides output (4.1 ± 0.5g/L).
The tolerance of different pyrimidine nucleoside analog, compared with starting strain TD131, is significantly improved: critical 100% lethal concentration of TD131 is respectively 150mg/L, 100mg/L and 80 μ g/L by 2-thiouracil, 6-azauracil and 5 FU 5 fluorouracil by the bacillus subtilis mutant strain of production pyrimidine nucleoside 3, provided by the present invention; Critical 100% lethal concentration of A219 is then brought up to 3g/L, 3.5g/L and 100mg/L by 2-thiouracil, 6-azauracil and 5 FU 5 fluorouracil respectively. The tolerance of 2-thiouracil is improve about 20 times; The tolerance of 6-azauracil is improve about 35 respectively; The tolerance of 5 FU 5 fluorouracil is improve about 125 times.
Accompanying drawing illustrates:
Figure 196 screening micropore plate result
Fig. 2 shaking flask the selection result
The upgrowth situation of thalline in Figure 35 L tank fermenting process
The consumption of glucose in Figure 45 L tank fermenting process
Figure 55 L tank fermenting process is urinated the accumulation volume of glycosides
Embodiment:
The screening of embodiment 1. subtilis A219
Initial bacterial strain TD131 is sieved again by the present invention through atmospheric pressure at room plasma mutagenesis-analog resistance screening-high flux screening-shaking flask, obtains strains A 219, and detailed process is as follows:
1. starting strain: B.subtilisTD131
2. atmospheric pressure at room plasma mutagenesis
(1) yeast culture method: protect tube and connect LB flat board, three zoning lines, cultivates 12h for 37 DEG C; From flat board, choose single bacterium colony meet LB and shake pipe, 37 DEG C, 200rpm, cultivate 12h; Shaking pipe switching LB shaking flask, inoculum size is 1%, 37 DEG C, 200rpm, cultivates 4-6h.
(2) preparation method of bacteria suspension: the thalline that centrifugal collection is cultivated, after stroke-physiological saline solution washing 2-3 time, then dilutes in right amount by stroke-physiological saline solution and makes OD600Being worth at the bacteria suspension of 0.6-0.8, getting 10 μ L bacteria suspensions, to be coated on slide glass pending.
(3) ARTP mutagenesis: ARTP processes parameter: slide glass is in flow ports 2mm place; Power is 120W; Airshed is 10SLM; Action time is 25s.
3. the screening of Analogue resistant mutant
Being coated on the minimum medium flat board containing finite concentration analog by bacteria suspension after ARTP mutagenic treatment, after 37 DEG C of cultivation 24h, the bacterial strain that bacterium colony is bigger is possible targeted mutagenesis bacterial strain.
4. high flux screening pyrimidine nucleoside high yield bacterium
Choosing analog resistant panel and get big bacterium colony point and receive on the LB flat board carrying out mark, and transfer sterilizing and fill in the deep hole of 400 Al sterile LB substratum, after the dull and stereotyped 37 DEG C of incubated overnight of LB, 4 DEG C of preservations, use as further screening. Dull and stereotyped 36 DEG C, 200rpm, after cultivating 10h, with the inoculum size of 10%, turns one by one to be inoculated into and fills in 400 micro-aseptic new orifice plates rising orifice plate fermention medium, and 36 DEG C of 3200rmp, cultivate 30h. After cultivation terminates, centrifugal fermented sample, after supernatant dilutes 100 times, gets 200 respectively and micro-is raised in enzyme plate, detects the light absorption value OD of sample under 270nm by microplate reader270, by OD270Higher bacterial strain carries out preservation, carries out further shaking flask and sieves again. Fig. 1 is screening micropore plate result.
5. shaking flask is sieved again
(1) substratum:
Activated inclined plane substratum (g/L): glucose 1, peptone 10, extractum carnis 10, yeast powder 5, NaCl2.5, agar 20, pH6.7～7.0,0.1MPa, 20min.
Seed culture medium (g/L): glucose 40, yeast powder 5, peptone 10, SODIUMNITRATE 15, potassium primary phosphate 1, dipotassium hydrogen phosphate 5.2, magnesium sulfate 1, Trisodium Citrate 1, pH6.7～7.0,0.075MPa, 15min.
Fermention medium (g/L): glucose 100, yeast powder 5, peptone 10, SODIUMNITRATE 15, potassium primary phosphate 1, dipotassium hydrogen phosphate 5.2, magnesium sulfate 1, Trisodium Citrate 1, pH6.7～7.0,0.075MPa, 15min. Grape sugar disappears.
(2) cultural method:
Slant culture: get-80 DEG C of preservation bacterial classification streak inoculations in activated inclined plane, cultivates 12h, and goes down to posterity once for 37 DEG C.
Seed culture: scrape a ring inclined-plane seed with transfering loop and be inoculated in the 500mL triangular flask that 30mL seed culture medium is housed, nine layers of gauze sealing, 37 DEG C, 200r/min shaking culture 6h.
Fermentation culture: draw 3mL seed liquor and be inoculated in the 500mL baffle flask that 27mL fermention medium is housed, nine layers of gauze sealing, 37 DEG C, 200r/min shaking culture 30h.
(3) analytical procedure:
PH measures: adopt pH6.4-8.0 accurate pH test paper to measure.
Biomass estimation: in fermenting process, with the absorbance OD600 at spectrophotometric determination 600nm place after suitably being diluted by fermented liquid, characterizes thalli growth situation.
Residual glucose: by fermented liquid in the centrifugal 2min of 13000r/min under room temperature, getting supernatant liquor 10 μ L, to be added in 990 μ L deionized waters fully mixed even, then getting 25 μ L adopts SBA-40E series of biologic sensing assays instrument to measure its residual sugar amount, institute's measured value is the residual sugar amount in fermented liquid, and unit is g/L.
Pyrimidine nucleoside concentration determination: get centrifugal after fermented liquid supernatant liquid 100 μ L join in 900 μ L deionized waters, comparing taking 1g/L standard model as ginseng and make identical dilution process, whole sample adopts HPLC to measure pyrimidine nucleoside output in fermented liquid after 0.45 μm of membrane filtration. Adopt chromatographic column be PhenomenexGemini5uC18110A150*4.6mm, post temperature 30 DEG C, moving phase is acetonitrile: water=2:98, flow velocity 1mL/min, each sample feeding 10 μ L, analysis time 12min.
Fig. 2 is the result of shaking flask screening.
The fermentation checking of 6.5L fermentor tank.
Embodiment 2 mutant strain A219 fermentative production urine glycosides pyrimidine
(1) the present embodiment substratum used:
Activated inclined plane substratum (g/L): glucose 1, peptone 10, extractum carnis 10, yeast powder 5, NaCl2.5, agar 20, pH6.7～7.0,0.1MPa, 20min.
Seed culture medium (g/L): glucose 40, yeast powder 5, peptone 10, SODIUMNITRATE 15, potassium primary phosphate 1, dipotassium hydrogen phosphate 5.2, magnesium sulfate 1, Trisodium Citrate 1, pH6.7～7.0,0.075MPa, 15min. The independent sterilizing of glucose.
5L ferment tank substratum (g/L): glucose 50, yeast powder 5, corn steep liquor 20, SODIUMNITRATE 15, potassium primary phosphate 1, dipotassium hydrogen phosphate 5.2, magnesium sulfate 5, ammonium sulfate 5, Trisodium Citrate 10, Sodium Glutamate 10, calcium chloride 1, manganous sulfate 0.02, zinc sulfate 0.02, pH6.7～7.0, the independent sterilizing of corn steep liquor, the independent sterilizing of calcium salt, magnesium manganese zinc salt is made into mixing solutions sterilizing together, glucose is made into the 80% independent sterilizing of solution, the sterilizing together of all the other components, sterilising conditions corn steep liquor is 0.1MPa, 20min, all the other are 0.075MPa, 15min.
(2) cultural method:
Slant culture: get-80 DEG C of preservation bacterial classification streak inoculations in activated inclined plane, cultivates 12h, and goes down to posterity once for 37 DEG C.
Seed culture: scrape a ring inclined-plane seed with transfering loop and be inoculated in the 1L triangular flask that 100mL seed culture medium is housed, nine layers of gauze sealing, 37 DEG C, 200r/min shaking culture 6h.
Fermentation culture: fermentor tank holds 3L surely, inoculum size is 10%, and culture temperature is 36 DEG C, pH7.0, and molten oxygen controls at about 20-30%, and residual sugar controls about 1%, and fermentation period is 48h.
(3) analytical procedure:
PH measures: adopt pH6.4-8.0 accurate pH test paper and fermentor tank pH electrode to measure.
Biomass estimation: in fermenting process, with the absorbance OD600 at spectrophotometric determination 600nm place after suitably being diluted by fermented liquid, characterizes thalli growth situation.
Residual glucose: by fermented liquid in the centrifugal 2min of 13000r/min under room temperature, getting supernatant liquor 10 μ L, to be added in 990 μ L deionized waters fully mixed even, then getting 25 μ L adopts SBA-40E series of biologic sensing assays instrument to measure its residual sugar amount, institute's measured value is the residual sugar amount in fermented liquid, and unit is g/L.
Pyrimidine nucleoside determination of yield: get centrifugal after fermented liquid supernatant liquid 100 μ L join in 900 μ L deionized waters, comparing taking 1g/L standard model as ginseng and make identical dilution process, whole sample adopts HPLC to measure pyrimidine nucleoside output in fermented liquid after 0.45 μm of membrane filtration. Adopt chromatographic column be PhenomenexGemini5uC18110A150*4.6mm, post temperature 30 DEG C, moving phase is acetonitrile: water=2:98, flow velocity 1mL/min, each sample feeding 10 μ L, analysis time 12min.
(4) fermentation results:
Fig. 1 is the upgrowth situation of A219 thalline in fermenting process and the comparison diagram of starting strain TD131, Fig. 2 is the consumption of A219 glucose in fermenting process and the comparison diagram of starting strain TD131, does not have too big-difference from the growth of comparing result two strain bacterium and consumption sugar situation. Fig. 3 is that A219 urinates the accumulation volume of glycosides and the comparison diagram of starting strain TD131 in fermenting process, from comparing result, ferments 48 hours, 3.3 times of the urine glycosides output of A219 to be 13.5g/L3 be starting strain TD131 urinates glycosides output (4.1g/L).
The tolerance of different structure analogue is detected by embodiment 3 mutant strain A219
(1) detection method: get 10 micro-bacterium liquid that rise and meet LB from protecting tube and shake pipe, 37 DEG C, 200rpm, cultivate 10-12 hour, the thalline that centrifugal collection has been cultivated, after stroke-physiological saline solution washs 2-3 time, with stroke-physiological saline solution gradient dilution 100,000 times, then get 100 micro-bacterium liquid that rise to coat respectively containing, on different concns analog minimum medium flat board, each concentration three is parallel, compare as minimum medium flat board (not containing analog).After 37 DEG C of cultivation 24h, dull and stereotyped chief's colony number is added up, the height of how many reflection tolerances of colony number. A large amount of bacterium colony can be grown in certain time in high density resistant panel and illustrate that this bacterial strain is strong to the tolerance of this analog.
Minimum medium (g/L): ammonium sulfate 3, dipotassium hydrogen phosphate 1, hydrolyzed casein 3, VB1、VB3、VB5、VB13, VH each 0.1mg, MgSO4·7H2O0.1, ZnSO4·7H2O、MnSO4·7H2The each 2mg of O, each 0.1g of tryptophane, Threonine, methionine(Met), Isoleucine, glucose 10, agar 20, wherein glucose is mixed with the solution of 800g/L, 0.075MPa, 15min sterilizing, all the other 0.1MPa, 20min sterilizings.
(2) detected result:
Table 1: two strain bacterium are to the tolerance detected result of different pyrimidine nucleoside analog
From detected result A219 compared with starting strain TD131, the tolerance of 2-thiouracil is improve about 20 times respectively; The tolerance of 6-azauracil is improve about 35 times; The tolerance of 5 FU 5 fluorouracil is improve about 125 times.
Claims (4)
1. a carbamyl phosphate synthetase Dare base mutant, it is characterised in that, the aminoacid sequence of described mutant is as shown in sequence table SEQ IDNo.4.
2. the encoding gene of mutant described in claim 1, it is characterised in that, the nucleotide sequence of described gene is as shown in SEQIDNo.2.
3. a bacillus subtilis mutant strain, it is characterised in that, comprise mutant according to claim 1, it is specially subtilis (Bacillussubtilis) A219, deposit number is CGMCCNo.11774.
4. the method for mutant strain fermentative production pyrimidine nucleoside described in claim 3, it is characterized in that, it is specially: inoculum size is 10%, culture temperature is 34-36 DEG C, pH6.7-7.0, and molten oxygen controls at 20-30%, residual sugar controls at 0.5-1%, fermentation period 48h, after having fermented, urinates glycosides concentration and can reach 13.5 ± 1g/L in fermented liquid;
Fermention medium forms in g/L: glucose 50, yeast powder 5, corn steep liquor 20, SODIUMNITRATE 15, potassium primary phosphate 1, dipotassium hydrogen phosphate 5.2, magnesium sulfate 5, ammonium sulfate 5, Trisodium Citrate 10, Sodium Glutamate 10, calcium chloride 1, manganous sulfate 0.02, zinc sulfate 0.02, pH6.7～7.0, the independent sterilizing of corn steep liquor, the independent sterilizing of calcium salt, magnesium manganese zinc salt is made into mixing solutions sterilizing together, glucose is made into the 80% independent sterilizing of solution, the sterilizing together of all the other components, sterilising conditions corn steep liquor is 0.1MPa, 20min, all the other are 0.075MPa, 15min.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108486162A (en) * | 2017-09-29 | 2018-09-04 | 天津科技大学 | A kind of production method of uridine |
| CN111321103A (en) * | 2020-03-17 | 2020-06-23 | 河南巨龙生物工程股份有限公司 | Escherichia coli mutant strain for high yield of cytidine and method for producing cytidine by fermentation |
| CN111411136A (en) * | 2019-09-27 | 2020-07-14 | 大连民族大学 | Preparation method for producing thymine by using marine Bacillus sp.JIN118 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108486162A (en) * | 2017-09-29 | 2018-09-04 | 天津科技大学 | A kind of production method of uridine |
| CN111411136A (en) * | 2019-09-27 | 2020-07-14 | 大连民族大学 | Preparation method for producing thymine by using marine Bacillus sp.JIN118 |
| CN111411136B (en) * | 2019-09-27 | 2023-05-12 | 大连民族大学 | Preparation method for producing thymine by Bacillus sp.JIN118 |
| CN111321103A (en) * | 2020-03-17 | 2020-06-23 | 河南巨龙生物工程股份有限公司 | Escherichia coli mutant strain for high yield of cytidine and method for producing cytidine by fermentation |
| CN111321103B (en) * | 2020-03-17 | 2021-12-31 | 河南巨龙生物工程股份有限公司 | Escherichia coli mutant strain for high yield of cytidine and method for producing cytidine by fermentation |
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