CN102994539A - Method for enhancing expression of corynebacterium crenatum NAD kinase to improve production capacity of strain L-arginine under high-low oxygen supply conditions - Google Patents
Method for enhancing expression of corynebacterium crenatum NAD kinase to improve production capacity of strain L-arginine under high-low oxygen supply conditions Download PDFInfo
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Abstract
The invention aims at enhancing the expression of corynebacterium crenatum NAD kinase to improve the production capacity of strain L-arginine. Under a high oxygen supply condition, the activity of NAD kinase is improved by 86.2%, NADP+ and NADPH are improved by 7.30% and 36.84% respectively, and the output of L-arginine is improved by 12%; and under a low-oxygen supply condition, the activity of NAD kinase is improved by 34.8%, the NADP+ and NADPH are improved by 14.67% and 15.0% respectively, and the output of L-arginine is improved by 38.5%.
Description
Technical field
The invention belongs to gene engineering technology field, be specifically related to a kind of reinforcement Corynebacterium crenatum NAD kinase expression, under the height oxygen supply condition, improve the method for this bacterial strain L-arginine throughput.
Background technology
L-arginine (L-Arginine, be called for short L-Arg) as a kind of semi-dispensable amino acid, it is the important source material of synthetic protein, phosphocreatine, it also is the important intermediate of ornithine cycle in the organism, have important physiological function and using value, be widely used in food, chemical industry and medicine and other fields.Because arginine has special using value, so the domestic needs amount is huge, adopt the fermentative Production L-arginine to have environmental protection, economic dispatch advantage, so make up high yield L-arginine engineering strain for realizing that fermentative Production L-arginine suitability for industrialized production is significant.
NADPH is a kind of important coenzyme in the organism, and as electron donor, it provides also prime mover for redox reaction, and plays an important role for the murder by poisoning of cell activity resistent oxygen.The kinase catalytic NAD of NAD
+Generate NADP with the ATP reaction
+With ADP, this is NADP
+Biosynthetic final step also is rate-limiting step, and NADP
+Generate NADPH by reduction reaction, it brings into play important physiological function in vivo.Corynebacterium glutamicum is as amino acid preparation strain, and the various amino acid requirements of its fermentative production carry out under the environment of high dissolved oxygen, provides sufficient NADPH significant for alleviating the environment that the residing oxygen of thalline coerces.NADPH has played vital role as the coenzyme in some redox reactions in the amino acid bio building-up process.In the L-arginine biosynthetic pathway, need the participation of NADPH: (1) by α-ketoglutaric acid through glutamate dehydrogenase (
Gdh) synthetic Pidolidone need a part NADPH; (2) need a part NADPH by N-acetylglutamat phosphoric acid through the synthetic N-acetylglutamat of acetylglutamate half aldehyde dehydrogenase-5-semialdehyde, so it is significant for improving its L-arginine throughput to increase thalline production NADPH ability.NADP in Corynebacterium glutamicum
+Reduction mainly realize by desaturase catalysis, wherein mainly by the glucose-6-phosphate dehydrogenase (G6PD) (G6PDH/ in the phosphopentose pathway (PPP approach)
Zwf) and 6-phosphogluconate dehydrogenase (6PGDH/
Gnd) provide, sub-fraction is by the isocitric enzyme (ICD/ in the TCA circulation in addition
Icd), malic enzyme (ME/
MalE), the NADPH that is wherein provided by G6PDH accounts for more than 70% of cell total amount, and G6PDH is the rate-limiting enzyme in the PPP approach, and the metabolism stream of this approach is subject to the regulation and control of [NADP+]/[NADPH] ratio.So increase NADP by strengthening the kinase whose expression of NAD
+Content, thereby the flow of regulation and control PPP approach, and then the content of NADPH is effective means in the increase born of the same parents.
Improving bacterial strain by the expression of strengthening enzyme or destructive enzyme gene is the main flow means of metabolic engineering for the throughput of target product, in addition, some researchs change bacterial strain to the throughput of target product by the level that changes coenzyme (such as NADPH), and are applied in intestinal bacteria, yeast saccharomyces cerevisiae, the Corynebacterium glutamicum.NADPH is as cofactors important in the amino acid biosynthetic pathway, increase the supply of NADPH by using expression that the metabolic engineering means strengthen the PPP approach or strengthen the synthetic genes involved of NADPH, have positive effect for improving bacterial strain amino acids production ability.According to pertinent literature, but the throughput of the Effective Raise bacterial strain 1Bs such as genes involved that in Corynebacterium glutamicum, synthesize about NADPH in logical disappearance glucosephosphate isomerase, the expression of strengthening G6PDH, the clonal expression intestinal bacteria.
Corynebacterium crenatum (
Corynebacterium crenatum) be that China investigator separates a strain corynebacterium, the gram positive bacterial strain that obtains.Its mutant strain is widely used in the amino acid industry at home.Corynebacterium crenatum SYPA is the strain L-arginine superior strain that this laboratory obtains by the multistage composite mutagenesis screening, and its condition bottom fermentation in high oxygen supply produces L-arginine.As the bacterial strain of plant height product L-arginine, it needs a large amount of NADPH that also prime mover is provided, and needing simultaneously sufficient NADPH to alleviate oxyradical as electron donor may be to the damaging action of microorganisms.The present invention increases the supply of NADPH in the born of the same parents by adding the biosynthetic rate-limiting enzyme of strongly expressed NADPH (NAD kinases), guarantee that on the one hand sufficient NADPH provides also prime mover for the L-arginine biosynthesizing; Alleviating on the other hand high oxygen supply fermentation condition may coerce the oxygen that bacterial strain produces.Increase by this method the ability that Corynebacterium crenatum produces L-arginine.
Summary of the invention
The invention provides the method that under height dissolved oxygen condition, improves this bacterial strain L-arginine throughput by strengthening Corynebacterium crenatum NAD kinase expression.Amplification coding NAD is kinase whose from Corynebacterium crenatum SYPA
PpnKGene and contains
TacThe shuttle expression plasmid pJC-of promotor
TacConnect, electric shock is transformed among the Corynebacterium crenatum SYPA, makes up recombinant bacterial strain, strengthens the kinase whose expression amount of NAD of this bacterial strain, thereby increases NADP in the born of the same parents
+And the content of NADPH, and then the output of raising L-arginine.Concrete invention scheme is as follows:
1. recombinant bacterial strain SYPA/pJC-
Tac-
PpnKObtain
On the clone Corynebacterium crenatum SYPA genome
PpnKGene, construction expression plasmid pJC-
Tac-
PpnK, this recombinant shuttle plasmid is changed in the Corynebacterium crenatum by the method that electric shock transforms, obtain recombinant corynebacterium crematum SYPA/pJC-
Tac-
PpnK
(1) shuttle plasmid pJC-
Tac-
PpnKThe structure of plasmid
Kinase whose according to coding NAD in the Corynebacterium glutamicum of announcing among the NCBI
PpnKGene order (GenBank:NC_006958), utilize its homology, in the design primer amplification Corynebacterium crenatum
PpnKGene.Design of primers is as follows:
ppnK?F?Xba?I：5’-GCTCTAGAATGACTGCACCCACGAACG-3’
ppnK?R?Sal?I：5’-GCGTCGACTTACCCCGCTGACCTGG-3’
Take the Corynebacterium crenatum genome as template, with
PpnKF reaches
PpnKR is primer, and amplification two ends restriction enzyme site is respectively Xbal I and Sal I
PpnKGene fragment, the PCR product is connected with pMD 19-T vector through behind the purifying, changes E. over to
ColiPreserve plasmid among the JM 109.Picking list bacterium colony from the flat board that contains Amp extracts plasmid and cuts checking through single, double enzyme, and order-checking.The plasmid that is connected with goal gene for success is with pJC-
TacCarry out double digestion with the same restrictions restriction endonuclease, purified rear connection obtains the pJC-tac-ppnk recombinant plasmid.
(2) recombinant bacterial strain SYPA/pJC-
Tac-
PpnKStructure
Extract plasmid pJC-
Tac-
PpnK, the method that transforms by electric shock is transformed in the Corynebacterium crenatum, coats on the Kan flat board that contains 50ug/L, cultivates 36h under 30 ℃ of conditions, and the picking transformant after cultivating, extracts the plasmid checking in the liquid nutrient medium that contains Kan.
2. the recombinant bacterial strain fermentation is estimated
Adopt the shaking flask of 250mL that Corynebacterium crenatum is carried out fermentation culture, set two different oxygen-supplying model, be that liquid amount is respectively 20mL, 60mL/250mL, investigate respectively under the different oxygen supply conditions, the difference of recombinant bacterial strain and starting strain fermenting process parameters is estimated the NAD kinases and is strengthened the impact of expressing strain growth, L-arginine production and carbon source consumption.Fermentation process is as follows:
Shake-flask seed substratum (gL
-1): glucose 30, yeast powder 10, urea 1.5, (NH
4)
2SO
420, KH
2PO
41, MgSO
47H
2O 0.5.NaOH transfers pH7.0～7.2, liquid amount 30 mL/250 mL.115 ℃ of sterilization 7min
Medium of shaking flask fermentation (gL
-1): glucose 150, yeast powder 10, KH
2PO
41.5, (NH
4)
2SO
450, MnSO
4H
2O 0.02, MgSO
47H
2O 0.5, FeSO
47H
2O 0.02, and L-His 5.0 * 10
-4, vitamin H 8 * 10
-5, lightweight CaCO
330.NaOH transfers pH7.0 ~ 7.2, liquid amount 20 mL/250 mL or 60 mL/250 mL, 115 ℃ of sterilization 7 min.
Cultural method: will preserve the line of bacterial classification and sugar bouillon medium slant, in 30 ℃ of constant temperature biochemical cultivation cases, cultivate about 20-24h, picking one ring bacterium is inoculated in the shake-flask seed substratum from the inclined-plane, on 30 ℃ of reciprocating type shaking tables of 120rpm/min, cultivate 14-16h to OD562 and be about 15-18, inoculum size by 5% is inoculated in Medium of shaking flask fermentation, on 30 ℃ of reciprocating type shaking tables of 120rpm/min, cultivate 24-36h, add 5% glucose, cultivate altogether 96h to fermentation ends.
The fermentation parameter measuring method:
A. thalli growth: the HCl with 0.25M carries out suitable dilution to fermented sample, in spectrophotometric determination A
562
The B.L-arginine content is measured: measure according to slope opening reagent method.
C. glucose content is measured: measure according to the DNS method.
3. determine pJC-
Tac-
PpnKIn Corynebacterium crenatum, add strongly expressed.
By the kinase whose activity of NAD in contrast recombinant bacterial strain and the starting strain, determine by shuttle plasmid pJC-
TacCarry goal gene
PpnKEffective expression in recombinant bacterial strain.
Result of the present invention: recombinant bacterial strain and starting strain are measured the kinase whose activity of NAD at the condition bottom fermentation of height oxygen supply in its logarithmic growth later stage, have improved respectively 2 times (HOS) and 1.2 times (LOS).Measure L-arginine output in thalline biomass and the fermented liquid behind the fermentation 108h, the biomass of recombinant bacterium is that 27g/L is low than starting strain (32g/L) under the high oxygen supply condition, has improved 12% but L-arginine output is 28.85g/L than starting strain (25.35g/L); Under the condition of low oxygen supply, the biomass of recombinant bacterium is that 17g/L is low than starting strain (21g/L), but that L-arginine output is 10.87g/L is high by 38.5% than starting strain (7.85g/L).Simultaneously, the activity of glucose-6-phosphate dehydrogenase (G6PD) improves in the recombinant bacterial strain, NADP
+And the content of NADPH is improved NAD
+And the content of NADH all has decline.Comprehensive above data, pJC-
Tac-
PpnKSuccessful expression in Corynebacterium crenatum, and the kinase whose expression of NAD are produced L-arginine to bacterial strain and are had positive meaning.
Description of drawings
Fig. 1 recombinant plasmid pJC-
Tac-
PpnKThe synoptic diagram that makes up
Fig. 2 pJC-
Tac-
PpnKNucleic acid electrophoresis Fig. 1 of plasmid construction: DL5000 marker 2:PCR amplification
PpnKProduct 3:pJC-
TacPlasmid enzyme restriction 4:pJC-
Tac-
PpnKSingle endonuclease digestion 5:pJC-
Tac-
PpnKWith Xba I and Sal I double digestion 6:DNA λ Hind III marker
The whole-cell protein electrophoresis of Fig. 3 height oxygen supply condition recombinant bacterial strain and starting strain
M: albumen Marker
1: high oxygen supply starting strain 2: high oxygen supply recombinant bacterial strain 3: low oxygen supply starting strain 4: low oxygen supply recombinant bacterial strain;
Recombinant bacterial strain and starting strain fermenting process are measured and comparison under the high oxygen supply condition of Fig. 4
(A) thalline biomass (B) L-arginine output (C) glucose content
(▲) recombinant bacterial strain (◆) starting strain
Recombinant bacterial strain and starting strain fermenting process are measured and comparison under the low oxygen supply condition of Fig. 5
(A) thalline biomass (B) L-arginine output (C) glucose content
(▲) recombinant bacterial strain (◆) starting strain
Fig. 6 uses the total free aminoacids in the high-efficient liquid phase analysis fermented liquid
Embodiment
Embodiment 1Recombinant corynebacterium crematum SYPA/pJC-
Tac-
PpnKStructure
1. recombinant bacterial strain SYPA/pJC-
Tac-
PpnKObtain
On the clone Corynebacterium crenatum SYPA genome
PpnKGene, construction expression plasmid pJC-
Tac-
PpnK, this recombinant shuttle plasmid is changed in the Corynebacterium crenatum by the method that electric shock transforms, obtain recombinant corynebacterium crematum SYPA/pJC-
Tac-
PpnK
(1) shuttle plasmid pJC-
Tac-
PpnKThe structure of plasmid
Kinase whose according to coding NAD in the Corynebacterium glutamicum of announcing among the NCBI
PpnKGene order, utilize its homology, in the design primer amplification Corynebacterium crenatum
PpnKGene.Design of primers is as follows:
ppnK?F?Xba?I：5’-GCTCTAGAATGACTGCACCCACGAACG-3’
ppnK?R?Sal?I：5’-GCGTCGACTTACCCCGCTGACCTGG-3’
Take the Corynebacterium crenatum genome as template, with
PpnKF reaches
PpnKR is primer, and amplification two ends restriction enzyme site is respectively Xbal I and Sal I
PpnKGene fragment, the PCR product is connected with pMD 19-T vector through behind the purifying, changes E. over to
ColiPreserve plasmid among the JM 109.Picking list bacterium colony from the flat board that contains Amp extracts plasmid and cuts checking through single, double enzyme, and order-checking.Obtain the right-on pMD 19-T-of goal gene sequence
PpnKPlasmid.
For the plasmid that successfully is connected into the T carrier, with pJC-
TacWith Xba I and Sal I double digestion, through nucleic acid electrophoresis respectively to goal gene fragment and pJC-
TacThe endonuclease bamhi recovery of tapping rubber is transformed into E. after connecting with T4 DNAligase
ColiPreserve plasmid among the JM 109.Picking list bacterium colony from the flat board that contains Kan extracts plasmid and verify through single double digestion, acquisition pJC-
Tac-
PpnKRecombinant plasmid.
Recombinant plasmid pJC-
Tac-
PpnKThe structure schematic flow sheet as shown in Figure 1, the nucleic acid electrophoresis figure of its building process is as shown in Figure 2.
PpnKThe about 960bp of gene is in Fig. 2
PpnKThe length of PCR product consistent.Afterwards recombinant plasmid is carried out single double digestion, about the about 7300bp of single endonuclease digestion pillar location, double digestion has two bands, and a position of cutting with the empty plasmid enzyme is suitable, one with
PpnKPCR product pillar location suitable, pJC-is described
Tac-
PpnKShuttle plasmid successfully constructs.
(2) recombinant bacterial strain SYPA/pJC-
Tac-
PpnKStructure
From E.
ColiExtract plasmid pJC-among the JM 109
Tac-
PpnK, the method that transforms by electric shock is transformed in the Corynebacterium crenatum competence, and electricity turns rear adding substratum, and jolting 2h on 30 ℃ of 120rpm/min shaking tables makes the thalline recovery, expresses resistant gene.Then bacterium liquid is coated on the Kan flat board that contains 50ug/L, cultivated 36h under 30 ℃ of conditions, the picking transformant after cultivating in the liquid nutrient medium that contains Kan, extracts plasmid and carries out PCR checking or double digestion checking.Select the bacterial strain that successfully transforms and carry out further fermenting experiment.
Embodiment 2:Recombinant bacterial strain and starting strain are investigated respectively the NAD kinase expression to the impact of its fermentation parameter respectively at the condition bottom fermentation of height oxygen supply.
Adopt the shaking flask of 250mL that Corynebacterium crenatum is carried out fermentation culture, set two kinds of different oxygen-supplying model, namely the shaking flask liquid amount is respectively 20/250mL and 60/250mL.Described in fermentation process such as the summary of the invention, fermentation 108h gets sample one time every 12h, measures the content of L-arginine and glucose in the dense and fermentation supernatant of the bacterium of all samples.Concrete Fermentation Process of Parameter such as Fig. 5.
As shown in Figure 5, under the environment of high oxygen supply, the thalline biomass of recombinant bacterial strain is lower than starting strain always, and this may be relevant with the larger recombinant plasmid of the carrier of recombinant bacterial strain own.To the 96h that ferments, the biomass of recombinant bacterium is that 27g/L reduces about 16% than starting strain (32g/L).In the process of L-arginine accumulation, recombinant bacterial strain does not show obvious advantage at earlier fermentation, and to the 72h that ferments, recombinant bacterial strain just shows the advantage of producing acid, to the 96h that ferments, recombinant bacterial strain L-arginine output is that 28.85g/L has improved 12% than starting strain (25.35g/L).In the consumption process of glucose, the wear rate of starting strain is higher than recombinant bacterial strain, and in the fermentation later stage, the gap of wear rate is dwindled, and the consumption of carbon source is mainly used in the breeding of thalline and the accumulation of L-arginine.
As shown in Figure 6, under the environment of low oxygen supply, the thalline biomass of recombinant bacterial strain is lower than starting strain always, and this situation with high oxygen supply is similar, but significantly decline under the environment of the higher oxygen supply of its thalline biomass.To the 96h that ferments, the biomass of recombinant bacterium is that 17g/L reduces about 19% than starting strain (21g/L).In the process of L-arginine accumulation, recombinant bacterial strain does not show obvious advantage at earlier fermentation, and to the 36h that ferments, the advantage of acid is produced in the recombinant bacterial strain performance, to the 96h that ferments, recombinant bacterial strain L-arginine output is that 10.87g/L has improved 38.5% than starting strain (7.85g/L).In the consumption process of glucose, be higher than recombinant bacterial strain at the wear rate of earlier fermentation starting strain, in the fermentation later stage, the sugar of recombinant bacterial strain consumption speed is obviously accelerated, and this may be because the raising of L-arginine accumulation volume is relevant.
By above fermenting experiment as seen, the NAD kinases strengthens expression to the growth of bacterial strain and the production existence impact of L-arginine under the condition of height oxygen supply.Improve to a certain extent the throughput of thalline L-arginine, had positive meaning so strengthen the kinase whose expression of NAD for Corynebacterium crenatum production L-arginine.
Embodiment 3Other free amino acid analysis in recombinant bacterium and the fermented liquid that sets out under the height oxygen supply condition
Described in the fermentation process such as summary of the invention of recombinant bacterium and the bacterium that sets out, to the 72h that ferments, take a sample under the height oxygen supply condition, centrifugal acquisition supernatant after suitably diluting, adopts the thiocarbanil derivatization method that the amino acid in the fermented sample is carried out derivatize.Sample after deriving is analyzed with high performance liquid chromatography, and the acetonitrile take 80% and sodium bicarbonate are moving phase, adopts the mode of gradient elution, analyzes collection of illustrative plates as shown in Figure 7.Heteroacid in the fermented liquid is less, and is the highest with L-arginine content, is Methionin, Isoleucine, glycine and phenylalanine etc. secondly.
Analytical results is as shown in table 3, wherein with arginine, Isoleucine and lysine content larger variation is arranged.Under height dissolved oxygen condition, the bacterium that sets out all has on the content of arginine, Methionin and Isoleucine in various degree and to improve recombinant bacterium.This may need NADPH to provide and prime mover is relevant with multiple redox reaction in the three seed amino acid biosynthetic pathways.So improving the method for the throughput of bacterial strain L-arginine by strengthening the kinase whose expression of NAD is effective.
Table 3 fermented liquid Analysis of Free Amino
Claims (4)
1. one kind is improved Corynebacterium crenatum SYPA produces L-arginine throughput under the height oxygen supply condition method, it is characterized by, by strengthening the NAD kinase expression of Corynebacterium crenatum SYPA, to improve the L-arginine throughput of this bacterial strain.
2. according to the method described in the claim 1, it is characterized by and use shuttle expression plasmid pJC-tac to carry coding NAD kinase whose
PpnKGene is expressed in Corynebacterium crenatum SYPA.
3. according to claim 1 described method, it is characterized by on the clone Corynebacterium crenatum SYPA genome
PpnKGene, construction expression plasmid pJC-
Tac-
PpnK, this recombinant shuttle plasmid is changed in the Corynebacterium crenatum by the method that electric shock transforms, obtain recombinant corynebacterium crematum SYPA/pJC-
Tac-
PpnK
(1) shuttle plasmid pJC-
Tac-
PpnKThe structure of plasmid
Kinase whose according to coding NAD in the Corynebacterium glutamicum of announcing among the NCBI
PpnKGene order, utilize its homology, in the design primer amplification Corynebacterium crenatum
PpnKGene, design of primers is as follows:
ppnK?FXba?I：5’-GC
TCTAGA ATGACTGCACCCACGAACG-3’
ppnK?R?SalI：5’-GC
GTCGAC TTACCCCGCTGACCTGG-3’
Take the Corynebacterium crenatum genome as template, with
PpnKF reaches
PpnKR is primer, and amplification two ends restriction enzyme site is respectively Xbal I and Sal I
PpnKGene fragment, the PCR product is connected with pMD 19-T vector through behind the purifying, changes E. over to
ColiPreserve plasmid among the JM 109; Picking list bacterium colony from the flat board that contains Amp extracts plasmid and cuts checking through single, double enzyme, and order-checking; The plasmid that is connected with goal gene for success is with pJC-
TacCarry out double digestion with the same restrictions restriction endonuclease, purified rear connection obtains pJC-
Tac-
PpnKRecombinant plasmid;
(2) recombinant bacterial strain SYPA/pJC-
Tac-
PpnKStructure
Extract plasmid pJC-
Tac-
PpnK, the method that transforms by electric shock is transformed in the Corynebacterium crenatum, coats on the Kan flat board that contains 50ug/L, cultivates 36h under 30 ℃ of conditions, and the picking transformant after cultivating, extracts the plasmid checking in the liquid nutrient medium that contains Kan.
4. according to claim 1 described method, it is characterized by and adopt the 250mL shaking flask to the recombinant corynebacterium crematum fermentation culture, make up two kinds of different oxygen supply patterns, liquid amount 20ml's is high oxygen environment, liquid amount 60ml's is low oxygen environment, utilizes respectively recombinant corynebacterium crematum to produce L-arginine under the yeasting of high oxygen supply and low oxygen supply.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103320438A (en) * | 2013-05-24 | 2013-09-25 | 江南大学 | Screening method for corynebacterium crenatum dissolved oxygen inducible promoter |
| CN103981231A (en) * | 2014-06-05 | 2014-08-13 | 江南大学 | Optimized high-yield technique for producing L-arginine Corynebacterium crenatum by batch fermentation |
| CN104531747A (en) * | 2014-12-09 | 2015-04-22 | 江南大学 | Method for improving L-arginine yield of corynebacterium crenatum by introducing poly-beta-hydroxybutyrate metabolic pathway |
| CN105018515A (en) * | 2015-07-15 | 2015-11-04 | 江西师范大学 | Method for improving yield of arginine by utilizing corynebacterium glutamicum and corynebacterium crenatum |
| CN106065411A (en) * | 2016-08-10 | 2016-11-02 | 洛阳华荣生物技术有限公司 | Fermentative Production creatine |
| CN106755210A (en) * | 2016-12-12 | 2017-05-31 | 安徽翠鸟生物技术有限公司 | Coenzyme mixed solution and preparation method thereof |
| CN110643587A (en) * | 2019-10-29 | 2020-01-03 | 杭州唯泰生物药业有限公司 | Method for preparing nicotinamide adenine dinucleotide phosphate by enzyme method |
| CN114751966A (en) * | 2021-12-30 | 2022-07-15 | 宁夏伊品生物科技股份有限公司 | Application of YH66_04585 protein and related biological materials thereof in improving arginine yield |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101381698A (en) * | 2008-10-23 | 2009-03-11 | 江南大学 | Recombinant corynebacterium crenatum for over expression of N-acetylglutamate kinase and application thereof |
| CN101397548A (en) * | 2008-10-23 | 2009-04-01 | 江南大学 | Recombinant corynebacterium crematum by expression of vitreoscilla haemoglobin gene and use thereof |
-
2012
- 2012-12-04 CN CN2012105119925A patent/CN102994539A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101381698A (en) * | 2008-10-23 | 2009-03-11 | 江南大学 | Recombinant corynebacterium crenatum for over expression of N-acetylglutamate kinase and application thereof |
| CN101397548A (en) * | 2008-10-23 | 2009-04-01 | 江南大学 | Recombinant corynebacterium crematum by expression of vitreoscilla haemoglobin gene and use thereof |
Non-Patent Citations (2)
| Title |
|---|
| 陶帅 等: "高产精氨酸的钝齿棒杆菌在高低供氧条件下的差异蛋白质组学初步研究", 《化工学报》 * |
| 陶帅: "高产L-精氨酸的钝齿棒杆菌在不同供氧条件的差异蛋白质组学研究", 《中国优秀硕士学位论文全文数据库工程科技I辑》 * |
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| CN103320438A (en) * | 2013-05-24 | 2013-09-25 | 江南大学 | Screening method for corynebacterium crenatum dissolved oxygen inducible promoter |
| CN103320438B (en) * | 2013-05-24 | 2015-06-24 | 江南大学 | Screening method for corynebacterium crenatum dissolved oxygen inducible promoter |
| CN103981231A (en) * | 2014-06-05 | 2014-08-13 | 江南大学 | Optimized high-yield technique for producing L-arginine Corynebacterium crenatum by batch fermentation |
| CN103981231B (en) * | 2014-06-05 | 2016-08-24 | 江南大学 | A kind of high yield L-arginine Corynebacterium crenatum batch fermentation optimizes technique |
| CN104531747A (en) * | 2014-12-09 | 2015-04-22 | 江南大学 | Method for improving L-arginine yield of corynebacterium crenatum by introducing poly-beta-hydroxybutyrate metabolic pathway |
| CN104531747B (en) * | 2014-12-09 | 2018-02-02 | 江南大学 | A kind of method for improving Corynebacterium crenatum L arginine yields by introducing poly beta-hydroxybutyrate metabolic pathway |
| CN105018515A (en) * | 2015-07-15 | 2015-11-04 | 江西师范大学 | Method for improving yield of arginine by utilizing corynebacterium glutamicum and corynebacterium crenatum |
| CN106065411A (en) * | 2016-08-10 | 2016-11-02 | 洛阳华荣生物技术有限公司 | Fermentative Production creatine |
| CN106755210A (en) * | 2016-12-12 | 2017-05-31 | 安徽翠鸟生物技术有限公司 | Coenzyme mixed solution and preparation method thereof |
| CN110643587A (en) * | 2019-10-29 | 2020-01-03 | 杭州唯泰生物药业有限公司 | Method for preparing nicotinamide adenine dinucleotide phosphate by enzyme method |
| CN114751966A (en) * | 2021-12-30 | 2022-07-15 | 宁夏伊品生物科技股份有限公司 | Application of YH66_04585 protein and related biological materials thereof in improving arginine yield |
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