CN109971676A - A kind of selection of the corynebacterium glutamicum of high yield isoleucine and application - Google Patents
A kind of selection of the corynebacterium glutamicum of high yield isoleucine and application Download PDFInfo
- Publication number
- CN109971676A CN109971676A CN201910223068.9A CN201910223068A CN109971676A CN 109971676 A CN109971676 A CN 109971676A CN 201910223068 A CN201910223068 A CN 201910223068A CN 109971676 A CN109971676 A CN 109971676A
- Authority
- CN
- China
- Prior art keywords
- corynebacterium glutamicum
- isoleucine
- strain
- high yield
- selection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
-
- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
Landscapes
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention provides a kind of corynebacterium glutamicum of fast high-flux breeding high-yield isoleucine (Corynebacteriumglutamicum) and application method B1, using Corynebacterium glutamicum as object, at 30-37 DEG C, it is cultivated in the shaking table of 100-200 r/min after a certain period of time, after normal temperature and pressure plasma (ARTP) mutagenic treatment 50-180 s, 30-37 DEG C culture 1-2 days in the sulphaguanidine culture medium of 0.05-0.4 mg/mL concentration are inoculated into, after culture, 96 orifice plate high flux screenings are carried out with the specific reaction of ninhydrin with corresponding strain fermentation supernatant using amino acid, superior strain can be selectedCorynebacteriumglutamicumB1.Bacterial strain isoleucine yield under conditions of 100mL shake flask fermentation 30-48 h compared with original strain improves 62.03%, and stabilization characteristics of genetics.This method establishes the high flux screening strategy of sulphaguanidine label primary dcreening operation and the detection of ninhydrin porous plate, can be widely applied to the corynebacterium glutamicum high throughput breeding of high yield isoleucine, improves screening efficiency, has huge application value.
Description
Technical field
The present invention relates to a kind of high-throughput screening method of high yield amino acid, especially a kind of glutamic acid rod of isoleucine
Bacillus high-throughput screening method belongs to biological medicine and big health industry.
Background technique
L-Isoleucine is one of eight kinds of essential amino acids of human body, while being one of three kinds of branched-chain amino acids, Yin Qite again
Different structure and function has especially important status in human life's metabolism.Therefore, it has been widely used in food, has moved
The industries such as object feed and medicine.The production method of l-Isoleucine mainly has protein Hydrolyze method, chemical synthesis and biology hair
Ferment method.Biological fermentation process is because its cost of material is low, prefered method of the easily controllable, energy conservation and environmental protection as industrialized production.Currently,
The yield of domestic isoleucine is not also high, and production technology is relatively external also to have larger gap, and the breeding of superior strain becomes different
The important link of leucine output increased.
Normal temperature and pressure plasma (ARTP) mutation breeding technologies are a kind of microorganism bases by Tsinghua University's independent research
Because of a group rapid mutation technology.The technology has at low cost, easy to operate, highly-safe and plasma Production conditions mildly rich
Containing a large amount of active particles, the spectrum of mutation is wide, mutation rate is high the advantages that, be widely used in the biology of the microorganisms such as bacterium, fungi, microalgae
Breeding.
Summary of the invention
In view of the above-mentioned problems, the present invention is using ARTP mutagenesis instrument to Corynebacterium glutamicum
(Corynebacteriumglutamicum) it is handled, primary dcreening operation is then marked by the sulfaguanidine resistance of addition suitable concentration,
Supernatant will be taken to carry out the detection of ninhydrin chromogenic reaction after primary dcreening operation strain inoculated to porous plate fermented and cultured again, realize high-throughput sieve
Choosing, at home and abroad has not been reported.
This technology goal of the invention is to provide the corynebacterium glutamicum of high yield isoleucine, Corynebacterium glutamicum B1, preservation
Number: CCTCC NO:M 2019118, classification naming Corynebacterium glutamicum B1, preservation date: 2019
March 1, preservation place: Hubei, Wuhan, Wuhan University, depositary institution: China typical culture collection center.The present invention establishes
A set of high flux screening process, superior strain ARTP mutagenesis screening key step: after original strain culture, first carries out ARTP
Then the optimization of mutagenic condition is marked by sulfaguanidine resistance and carries out primary dcreening operation, selected the full bacterium colony of abundance, be inoculated into porous plate
Middle culture is extracted fermented supernatant fluid and is reacted under certain condition with ninhydrin reagent, picks out in OD570Lower light absorption value is biggish
The corresponding fermented supernatant fluid of bacterial strain[7], thin layer chromatography specific aim control test is carried out, then through shaking flask secondary screening culture, extract supernatant
Liquid detects its isoleucine content using amino-acid analyzer.The breeding side of the Corynebacterium glutamicum of specific high yield isoleucine
Method includes the following steps that process is shown in Fig. 1.
(1) it using Corynebacterium glutamicum as object, is cultivated in 30-37 DEG C, the shaking table of 100-200r/min a period of time, then
By normal temperature and pressure plasma mutagenic treatment 30-180s；
(2) be inoculated into sulphaguanidine culture medium after processing to be mutagenic, 30-37 DEG C culture 1-2 days；
(3) dominant strain is identified after the bacterial strain that step (2) obtains being carried out specific chromogenic reaction using ninhydrin, it will
After the dominant strain is cultivated in seed culture medium, then cultivate in the fermentation medium, after carrying out amino acid analysis detection secondary screening,
Screening obtains the highest bacterial strain Corynebacterium glutamicumB1 of yield.
The radiant power of the normal temperature and pressure plasma mutagenesis is 100-120W, and helium gas flow 3-10L/min is lured
The change time is 120-150s.
Sulphaguanidine containing 0.05-0.4mg/mL in the sulphaguanidine culture medium.
Bacterial strain is reacted with ninhydrin using porous plate progress specific chromogenic in the step (3), and porous plate includes 48-
96 orifice plates.The dominant strain by porous plate first in seed culture medium, is cultivated at 30-37 DEG C, 100-200r/min
1-2 days；It is seeded in fermentation medium with the inoculum concentration of 5-10% again, then cultivates 1-2 at 30-37 DEG C, 100-200r/min
It, after carrying out amino acid analysis detection secondary screening, the stable superior strain of available performance
CorynebacteriumglutamicumB1。
The seed liquid culture medium g/L includes: glucose 5-10g/L, peptone 1-5g/L, sodium chloride 1-5g/L, ox
Meat soaks powder 1-3g/L, disodium hydrogen phosphate 0.03-1g/L；
Fermentation medium g/L includes: glucose 5-10g/L, peptone 2-7g/L, sodium chloride 1-5g/L, beef extract powder 1-
3g/L, disodium hydrogen phosphate 0.3-1g/L, yeast extract 1-5g/L, ammonium sulfate 1-6g/L.For the above-mentioned processing step present invention
The technical solution system thinking influence of the ARTP mutagenesis lethality of this method
Mutagenic treatment is carried out using the different time (0,60,90,120,150,180,210,240s), by the thallus of mutagenesis
It is put into the physiological saline of 1mL and shakes 1min, take 100uL to be applied on plating medium and cultivate 2 days for 37 DEG C, calculate lethal
Rate.
Studies have shown that the active particle in plasma acts on microorganism, microorganism wall/film structure can be made
And permeability changes, and cause gene damage, and then make microbial gene sequences and its metabolism network that significant changes occur, finally
Microorganism is caused to generate mutation.Mutation time and action intensity are affected to microorganism lethality.Lethality is too low or mistake
Height is all unfavorable for screening.As a result see as shown in Figure 2.
ARTP is larger to the lethality of Corynebacteriumglutamicum as can be seen from Figure 2,60-120s it
Between linear growth pattern, handle the time be 120s when lethality reach 88% or so, 180s when lethality reach
98.44%, when treated between when reach 240s, thallus is substantially all to survive.In order to guarantee certain mutation rate,
Select corresponding mutation time most important.Experiment show the corresponding radiated time for selecting lethality to reach 95% or more for
Preferably.
System thinking influence of the sulphaguanidine concentration to bacterial strain inhibiting rate is gone back in further this technology invention
Sulphaguanidine is the analogue of aspartic acid, solves day by breeding sulfaguanidine resistance mutant strain energy heredity
Feedback inhibition of the aspartic acid to phosphoric acid enol pyruvic acid carboxylase.Simultaneously 0,0.1,0.15,0.2,0.25,0.3,0.35,
The sulphaguanidine of the various concentrations such as 0.4mg/mL is different to the inhibition level of cell, it is therefore desirable to which optimizing sulphaguanidine concentration makes to screen
Effect reaches best.Different sulphaguanidine concentration is chosen to be studied.Different sulphaguanidine concentration pair
The inhibiting rate of Corynebacteriumglutamicum is as shown in Figure 3.
Sulphaguanidine is obvious to the inhibitory effect of Corynebacteriumglutamicum as can be seen from Figure 3.In low concentration
When (0.1mg/mL), inhibiting effect is smaller, and when sulphaguanidine concentration reaches 0.3mg/mL, inhibitory effect enhancing, inhibiting rate reaches
97% or so.When sulphaguanidine concentration reaches 0.4mg/mL, thallus cannot be grown.In order to while thallus can be enable to grow
Screening effect can be reached again, choose best addition of the sulphaguanidine concentration additive amount of 0.3mg/mL or so as screening and culturing medium
Amount is used for sulfaguanidine resistance label screening.
In addition, technical solution of the present invention has carried out the high flux screening of dominant strain
Since the mutant that mutagenesis generates is more, so needing to establish a kind of quick screening mode of high throughput.First by plate
The bacterial strain of upper mutagenesis is inoculated into the culture medium of 24 hole 1mL with bamboo stick, is trained in 37 DEG C, the microwell plate constant-temperature table of 300r/min
It supports, takes culture 24,36, the 20 μ L of bacterium solution centrifuging and taking supernatant of 48h into 96 circular hole deep-well plates, 240 μ are added in each orifice plate
The ninhydrin solution of L0.5%, 70 DEG C of water-bath 8min, then cools to room temperature, its absorbance is surveyed with microplate reader, not add
The absorption luminous intensity of ninhydrin solution is blank control, filters out the higher mutant strain of amino acid content.And mutagenic strain is existed
Continuous passage 10 times on plating medium, then fermented and cultured is carried out, the acid producing ability of bacterial strain is detected, its genetic stability is studied.
Fig. 4 is shown with original strain photon absorbing intensity when being 1.0, and opposite extinction when 42 mutagenic strain culture 48h is strong
Degree.As seen from the figure, the opposite photon absorbing intensity of most of mutagenic strains is higher than original strain, there is three plants of mutagenic strains B1, D6, E5
Opposite light absorption value is higher than 20% or more original strain, and mutagenic strain B1, D6, E5 and original strain W are carried out shake flask fermentation culture,
Isoleucine yield when 48h is detected using amino-acid analyzer.Secondary screening result such as table 1.
The result of 1 Corynebacterium glutamicum mutagenesis secondary screening of table
As can be seen from Table 1, it for the relatively primitive bacterial strain of shaking flask secondary screening yield of mutagenic strain B1, D6, E5, still shows
Preferable isoleucine production performance, yield have increased separately 62.03%, 30.89%, 14.19% out.It can be seen that this reality
The screening technique tested is effective.
Finally, the bacterial strain that technical solution of the present invention obtains has carried out genetic stability investigation
For the heredity spy for examining advantage mutagenic strain Corynebacterium glutamicumB1 that can keep stable
Property, by mutagenic strain B1 continuous passage culture 10 times, and fermented and cultured is carried out simultaneously, detect fermented supernatant fluid at 570nm
Photon absorbing intensity, as a result as shown in figure 5, from 1st generation to the photon absorbing intensity kept stable of the 10th generation bacterial strain, it is seen that the mutagenic bacteria
Strain has good genetic stability.
The screening technique that technical solution of the present invention establishes efficient high yield isoleucine bacterial strain is ARTP mutation breeding
Difficult point.In the synthesis path of isoleucine, there is feedback inhibition to phosphoric acid enol pyruvic acid carboxylase for aspartic acid
Effect, can make metabolic fluxes more unobstructed, to increase the yield of isoleucine if releasing the feedback inhibition.Sulphaguanidine is asparagus fern ammonia
The analogue of acid, if sulphaguanidine to be added in culture medium to the screening for being used for bacterial strain, it is expected to obtain sulfaguanidine resistance mutation
Strain, the genetic releasing aspartic acid of energy keep aspartic acid a large amount of the feedback inhibition of phosphoric acid enol pyruvic acid carboxylase
Synthesis.Using the chromogenic reaction of ninhydrin and amino acid, it can tentatively compare free aminoacid content in fermentation liquid, then in conjunction with more
Function microplate reader is expected to realize the high flux screening of porous plate.
The key enzyme in microorganism Acid production metabolism approach is inhibited to combine by orienting ARTP random mutagenesis with sulphaguanidine,
Increase the probability that mutagenic strain is mutated to isoleucine accumulation direction, increases the probability for screening strain excellent, reduce screening
Workload, provide efficient screening technique for the breeding of acid-producing bacteria strain, mentioned for the screenings of other high yield isoleucine bacterial strains
For important references.
Detailed description of the invention
Fig. 1 is the process flow chart of the Corynebacterium glutamicum high-throughput screening method of isoleucine.
Fig. 2 is Corynebacterium glutamicum ARTP lethality curve.
Fig. 3 is various concentration sulphaguanidine to Corynebacteriumglutamicum inhibiting rate curve.
Fig. 4 is the opposite photon absorbing intensity of mutant strain.
The photon absorbing intensity that Fig. 5 is mutagenic strain continuous passage ten times.
Specific embodiment
Embodiment 1
It in radiant power is 110W, helium by ARTP from original strain Corynebacteriumglutamicum
After flow is 8L/min mutagenic treatment 130s, it is inoculated into the sulphaguanidine culture medium of 0.2mg/mL concentration and cultivates 1 day for 34 DEG C, select
Then the full bacterial strain of abundance out is seeded to 96 orifice plate fluid nutrient medium cultures, the specificity using amino acid and ninhydrin is anti-
Should with corresponding strain fermentation supernatant carry out high flux screening, select the maximum bacterial strain of photon absorbing intensity, glucose 8g/L,
Peptone 3g/L, sodium chloride 2g/L, beef extract powder 2g/L, disodium hydrogen phosphate 0.04g/L seed culture medium be 35 DEG C lower, 180 r/
Min cultivates 36h, carries out seed culture, is then seeded to fermentation medium glucose 8g/L, albumen with the inoculum concentration of 8% (v/v)
In peptone 5g/L, sodium chloride 4g/L, beef extract powder 2g/L, disodium hydrogen phosphate 0.8g/L, yeast extract 4g/L, ammonium sulfate 5g/L in
35 DEG C, 180r/min culture 36h, after then carrying out amino acid analysis detection secondary screening result, the stable high yield of available performance
Bacterial strain Corynebacterium glutamicumB1 produces isoleucine than original strain and improves 55.6%.
Embodiment 2
It in radiant power is 100W, helium by ARTP from original strain Corynebacteriumglutamicum
After flow is 7L/min mutagenic treatment 150s, it is inoculated into the sulphaguanidine culture medium of 0.3mg/mL concentration and cultivates 1 day for 33 DEG C, select
Then the full bacterial strain of abundance out is seeded to 96 orifice plate fluid nutrient medium cultures, the specificity using amino acid and ninhydrin is anti-
Should with corresponding strain fermentation supernatant carry out high flux screening, select the maximum bacterial strain of photon absorbing intensity, glucose 8g/L,
In 34 DEG C, 150 r/ under peptone 3g/L, sodium chloride 2g/L, beef extract powder 2g/L, disodium hydrogen phosphate 0.04g/L seed culture medium
Min cultivates 1-2 days progress seed cultures, is then seeded to glucose 8g/L, peptone 5g/L, chlorine with the inoculum concentration of 7% (v/v)
In the fermentation medium for changing sodium 4g/L, beef extract powder 2g/L, disodium hydrogen phosphate 0.5g/L, yeast extract 4g/L, ammonium sulfate 5g/L
35 DEG C, 150r/min culture 40h, after carrying out amino acid analysis detection secondary screening, the stable superior strain of available performance
Corynebacterium glutamicumB1 produces isoleucine than original strain and improves 60.8%.
Embodiment 3
It in radiant power is 120W, helium by ARTP from original strain Corynebacteriumglutamicum
After flow is 3L/min mutagenic treatment 120s, it is inoculated into the sulphaguanidine culture medium of 0.08mg/mL concentration and cultivates 1 day for 30 DEG C, choose
The full bacterial strain of abundance is selected, 96 orifice plate fluid nutrient medium cultures is then seeded to, utilizes the specificity of amino acid and ninhydrin
Reaction carries out high flux screening with corresponding strain fermentation supernatant, the maximum bacterial strain of photon absorbing intensity is selected, in glucose 6g/
L, peptone 1g/L, sodium chloride 5g/L, beef extract powder 3g/L, disodium hydrogen phosphate 0.5g/L seed culture medium be 35 DEG C lower, 120r/
Min cultivates 36h, carries out seed culture, is then seeded to fermentation medium glucose 5g/L, egg with the inoculum concentration of 10% (v/v)
White peptone 2g/L, sodium chloride 2g/L, beef extract powder 1g/L, disodium hydrogen phosphate 0.2g/L, in yeast extract 2g/L, ammonium sulfate 3g/L
39h is cultivated in 30 DEG C, 150r/min, after then carrying out amino acid analysis detection secondary screening result, the stable height of available performance
Bacterial strain Corynebacterium glutamicumB1 is produced, isoleucine is produced than original strain and improves 61.7%.
Embodiment 4
It in radiant power is 100W, helium by ARTP from original strain Corynebacteriumglutamicum
After flow is 10L/min mutagenic treatment 140s, it is inoculated into the sulphaguanidine culture medium of 0.4mg/mL concentration and cultivates 2 days for 37 DEG C, choose
The full bacterial strain of abundance is selected, 96 orifice plate fluid nutrient medium cultures is then seeded to, utilizes the specificity of amino acid and ninhydrin
Reaction carries out high flux screening with corresponding strain fermentation supernatant, the maximum bacterial strain of photon absorbing intensity is selected, in glucose 10
Under g/L, peptone 5g/L, sodium chloride 1g/L, beef extract powder 1g/L, disodium hydrogen phosphate 0.07g/L seed culture medium in 34 DEG C,
200r/min cultivates 1-2 days progress seed cultures, is then seeded to glucose 10g/L, peptone 4 with the inoculum concentration of 5% (v/v)
The fermentation training of g/L, sodium chloride 5g/L, beef extract powder 3g/L, disodium hydrogen phosphate 0.6g/L, yeast extract 3g/L, ammonium sulfate 6g/L
37 DEG C, 170r/min culture 45h, after carrying out amino acid analysis detection secondary screening, the stable Producing Strain of available performance are supported in base
Strain Corynebacterium glutamicumB1 produces isoleucine than original strain and improves 63.5%.
Claims (7)
1. a kind of corynebacterium glutamicum of high yield isoleucine, Corynebacterium glutamicum B1, deposit number: CCTCC NO:M
2019118, classification namingCorynebacteriumglutamicumB1, preservation date: on March 16th, 2019, Hubei are military
The Chinese.
2. a kind of selection of the corynebacterium glutamicum of high yield isoleucine described in claim 1, which is characterized in that packet
Include following steps:
(1) using Corynebacterium glutamicum as object, a period of time is cultivated in 30-37 DEG C, the shaking table of 100-200 r/min, then pass through
Cross normal temperature and pressure plasma mutagenic treatment 30-180 s；
(2) be inoculated into sulphaguanidine culture medium after processing to be mutagenic, 30-37 DEG C culture 1-2 days；
(3) dominant strain is identified after the bacterial strain that step (2) obtains being carried out specific chromogenic reaction using ninhydrin, this is excellent
After gesture bacterial strain is cultivated in seed culture medium, then cultivate in the fermentation medium, after carrying out amino acid analysis detection secondary screening, screening
Obtain the highest bacterial strain of yield, the i.e. corynebacterium glutamicum of high yield isoleucineCorynebacteriumglutamicumB1。
3. the selection of the corynebacterium glutamicum of high yield isoleucine according to claim 2, which is characterized in that often
The radiant power of normal temperature and pressure plasma mutagenesis is 100-120 W, and helium gas flow is 3-10 L/min, mutation time 120-
150s。
4. the corynebacterium glutamicum selection of high yield isoleucine according to claim 2, which is characterized in that sulfanilamide (SN)
Sulphaguanidine containing 0.05-0.4 mg/mL in guanidine culture medium.
5. the Corynebacterium glutamicum selection of high yield isoleucine according to claim 2, which is characterized in that step
(3) bacterial strain is reacted with ninhydrin using porous plate progress specific chromogenic in, and porous plate includes 48-96 orifice plate.
6. the corynebacterium glutamicum selection of high yield isoleucine according to claim 5, which is characterized in that will be more
The dominant strain of orifice plate first in seed culture medium, is cultivated 1-2 days at 30-37 DEG C, 100-200 r/min；Again with 5-10%
Inoculum concentration be seeded in fermentation medium, then cultivated 1-2 days at 30-37 DEG C, 100-200 r/min, carry out Amino acid score
After analysis detection secondary screening, the stable superior strain of available performanceCorynebacteriumglutamicumB1。
7. the corynebacterium glutamicum selection of high yield isoleucine according to claim 6, which is characterized in that used
Seed liquid culture medium g/L include: glucose 5-10g/L, peptone 1-5g/L, sodium chloride 1-5g/L, beef extract powder 1-3g/L,
Disodium hydrogen phosphate 0.03-1g/L；
Fermentation medium g/L includes: glucose 5-10g/L, peptone 2-7g/L, sodium chloride 1-5g/L, beef extract powder 1-3g/L,
Disodium hydrogen phosphate 0.3-1g/L, yeast extract 1-5g/L, ammonium sulfate 1-6g/L.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210367357.8A CN114908079A (en) | 2019-03-22 | 2019-03-22 | Breeding method of corynebacterium glutamicum capable of producing isoleucine at high yield |
| CN201910223068.9A CN109971676B (en) | 2019-03-22 | 2019-03-22 | Breeding method and application of corynebacterium glutamicum capable of highly producing isoleucine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910223068.9A CN109971676B (en) | 2019-03-22 | 2019-03-22 | Breeding method and application of corynebacterium glutamicum capable of highly producing isoleucine |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210367357.8A Division CN114908079A (en) | 2019-03-22 | 2019-03-22 | Breeding method of corynebacterium glutamicum capable of producing isoleucine at high yield |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109971676A true CN109971676A (en) | 2019-07-05 |
| CN109971676B CN109971676B (en) | 2022-04-08 |
Family
ID=67080138
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210367357.8A Pending CN114908079A (en) | 2019-03-22 | 2019-03-22 | Breeding method of corynebacterium glutamicum capable of producing isoleucine at high yield |
| CN201910223068.9A Active CN109971676B (en) | 2019-03-22 | 2019-03-22 | Breeding method and application of corynebacterium glutamicum capable of highly producing isoleucine |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210367357.8A Pending CN114908079A (en) | 2019-03-22 | 2019-03-22 | Breeding method of corynebacterium glutamicum capable of producing isoleucine at high yield |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN114908079A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111197014A (en) * | 2020-04-01 | 2020-05-26 | 宜昌三峡普诺丁生物制药有限公司 | Corynebacterium glutamicum mutant strain and application thereof |
| CN112795607A (en) * | 2020-12-31 | 2021-05-14 | 河南巨龙生物工程股份有限公司 | Method for improving adenosine fermentation yield |
| CN114774403A (en) * | 2022-04-27 | 2022-07-22 | 湖州师范学院 | Targeting breeding method of agricultural lactic acid bacteria |
| CN116121135A (en) * | 2022-12-29 | 2023-05-16 | 大连医诺生物股份有限公司 | Corynebacterium glutamicum mutant strain and application thereof in L-isoleucine fermentation |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113444655B (en) * | 2020-03-26 | 2023-05-16 | 吉林中粮生化有限公司 | Corynebacterium glutamicum, temperature-sensitive strain with high glutamic acid yield, obtaining method and application thereof, and glutamic acid fermentation method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3009504A1 (en) * | 2013-06-11 | 2016-04-20 | Cj Cheiljedang Corporation | Microorganism producing l-isoleucine and method for preparing l-isoleucine using same |
| CN105886431A (en) * | 2016-04-27 | 2016-08-24 | 天津科技大学 | Corynebacterium glutamicum and method for producing high-yield isoleucine with same |
| CN108841758A (en) * | 2018-07-13 | 2018-11-20 | 大连医诺生物股份有限公司 | Corynebacterium glutamicum mutant and its application in L-Leu production |
| CN109576261A (en) * | 2019-01-23 | 2019-04-05 | 三峡大学 | A kind of selection of the Corynebacterium glutamicum of high yield isoleucine and application |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1314798C (en) * | 2005-06-06 | 2007-05-09 | 无锡晶海氨基酸有限公司 | L-leucine high-yield bacterium and fermentation method using the same for L-leucine production |
| KR101335789B1 (en) * | 2012-01-13 | 2013-12-02 | 씨제이제일제당 (주) | Microorganism producing L-isoleucine and process for preparing L-isoleucine using the same |
-
2019
- 2019-03-22 CN CN202210367357.8A patent/CN114908079A/en active Pending
- 2019-03-22 CN CN201910223068.9A patent/CN109971676B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3009504A1 (en) * | 2013-06-11 | 2016-04-20 | Cj Cheiljedang Corporation | Microorganism producing l-isoleucine and method for preparing l-isoleucine using same |
| CN105886431A (en) * | 2016-04-27 | 2016-08-24 | 天津科技大学 | Corynebacterium glutamicum and method for producing high-yield isoleucine with same |
| CN108841758A (en) * | 2018-07-13 | 2018-11-20 | 大连医诺生物股份有限公司 | Corynebacterium glutamicum mutant and its application in L-Leu production |
| CN109576261A (en) * | 2019-01-23 | 2019-04-05 | 三峡大学 | A kind of selection of the Corynebacterium glutamicum of high yield isoleucine and application |
Non-Patent Citations (5)
| Title |
|---|
| LIANGHONG YIN 等: "Enhancing the Carbon Flux and NADPH Supply to Increase L-isoleucine Production in Corynebacterium glutamicum", 《BIOTECHNOLOGY AND BIOPROCESS ENGINEERING》 * |
| XIAOYUAN WANG: "Strategy for improving L-isoleucine production eff iciency in Corynebacterium glutamicum", 《APPLIED MICROBIOLOGY AND BIOTECHNOLOGY》 * |
| 孔帅 等: "常温常压等离子体诱变选育高产L-异亮氨酸谷氨酸棒杆菌", 《中国酿造》 * |
| 王壮壮 等: "L-异亮氨酸高产菌选育及其培养基优化", 《生物技术通报》 * |
| 谢飞 等: "L-异亮氨酸高产菌种的选育研究", 《中国酿造》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111197014A (en) * | 2020-04-01 | 2020-05-26 | 宜昌三峡普诺丁生物制药有限公司 | Corynebacterium glutamicum mutant strain and application thereof |
| CN111197014B (en) * | 2020-04-01 | 2022-11-08 | 宜昌三峡普诺丁生物制药有限公司 | Corynebacterium glutamicum mutant strain and application thereof |
| CN112795607A (en) * | 2020-12-31 | 2021-05-14 | 河南巨龙生物工程股份有限公司 | Method for improving adenosine fermentation yield |
| CN112795607B (en) * | 2020-12-31 | 2023-06-23 | 河南巨龙生物工程股份有限公司 | Method for improving adenosine fermentation yield |
| CN114774403A (en) * | 2022-04-27 | 2022-07-22 | 湖州师范学院 | Targeting breeding method of agricultural lactic acid bacteria |
| CN114774403B (en) * | 2022-04-27 | 2023-08-08 | 至农科技发展(浙江)有限公司 | Targeted breeding method for agricultural lactic acid bacteria |
| CN116121135A (en) * | 2022-12-29 | 2023-05-16 | 大连医诺生物股份有限公司 | Corynebacterium glutamicum mutant strain and application thereof in L-isoleucine fermentation |
| CN116121135B (en) * | 2022-12-29 | 2024-04-09 | 大连医诺生物股份有限公司 | Corynebacterium glutamicum mutant strain and application thereof in L-isoleucine fermentation |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114908079A (en) | 2022-08-16 |
| CN109971676B (en) | 2022-04-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109971676A (en) | A kind of selection of the corynebacterium glutamicum of high yield isoleucine and application | |
| CN101724587B (en) | Lactobacillus brevis L2 bacterial strain of high yield gamma-aminobutyrique and screening method and applications thereof | |
| CN105886431A (en) | Corynebacterium glutamicum and method for producing high-yield isoleucine with same | |
| CN104388330B (en) | A kind of method of L tryptophans fermentation strain and its fermenting and producing L tryptophans | |
| CN109576261A (en) | A kind of selection of the Corynebacterium glutamicum of high yield isoleucine and application | |
| CN102703339B (en) | High-yield arginine deiminase bacterial strain and method for producing L-citrulline by same | |
| CN110373359A (en) | A kind of streptomyces albus X-18 and the method using bacterium production epsilon-polylysine | |
| CN104988082B (en) | The inferior Dbaly yeast bacterial strain of one plant of Chinese and its application | |
| CN103361289B (en) | Strain for producing L-lysine and method thereof for producing L-lysine | |
| CN109082448A (en) | A kind of Escherichia coli and its application in fermenting and producing 1,5- pentanediamine | |
| CN109609491A (en) | It is a kind of it is efficient produce kasugarnycin streptomyces microaureus mutagenesis and screening technique | |
| CN105969702B (en) | Serratia marcescens RZ 21-C6 and its application | |
| CN105820991B (en) | A kind of Recombinant organism | |
| CN106047740B (en) | One plant of l-Alanine superior strain | |
| CN104726381A (en) | Bacterial strain for producing L-lysine and L-lysine producing method thereof | |
| CN108642041B (en) | Method for improving L-alanine fermentation production capacity of recombinant escherichia coli | |
| CN105671008B (en) | The bacterial strain and its carbamyl phosphate synthetase regulatory site of one plant height production pyrimidine nucleoside | |
| CN101974452B (en) | Colimycin high-yield strain and screening method thereof | |
| CN106834181B (en) | A kind of Pediococcus acidilactici and its application | |
| CN102936575B (en) | Escherichia coli LL016 and application thereof | |
| CN107557311A (en) | One plant of lactobacillus acidophilus and its application in antibacterial polypeptide is produced in fermentation | |
| CN108728370A (en) | The salmon subfamily Renibacterium bacterial strain QD-01 and its fermentation process of one plant height effect production chitosan enzyme and application | |
| CN106282068A (en) | The preparation method of resistance controllable type bacillus spore | |
| KR100724699B1 (en) | Novel Corynebactrium glutamicum which is Used For Industrial Production of L-Valine and Manufacturing Method of L-Valine Using The Microorganism | |
| CN116790382B (en) | High-yield lutein mutant chlorella, and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20221223 Address after: 444100 Group 5, Shuifumiao Village, Longquan Town, Yiling District, Yichang City, Hubei Province Patentee after: Bee Production (Yichang) Biotechnology Co.,Ltd. Address before: 443002 No. 8, University Road, Xiling District, Yichang, Hubei Patentee before: CHINA THREE GORGES University |