CN109456961A - A kind of high yield YT-011A strain mutagenesis selection and its fermentation medium - Google Patents
A kind of high yield YT-011A strain mutagenesis selection and its fermentation medium Download PDFInfo
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Abstract
The present invention relates to a kind of high yield YT-011A strain mutagenesis selection, specifically: mutagenesis is iterated using ARTP-NT6 complex mutation to original strain, and is aided with streptomycin resistance breeding, obtains enhanced variant.Furthermore a kind of fermentation medium of optimization is provided for above-mentioned gained enhanced variant.Using ARTP-NTG iteration complex mutation; it is screened in conjunction with streptomycin resistance; the mutagenic strain of the high yield YT-011A of acquisition; the yield increase rate of mutagenic strain generally reaches 50% or more; wherein YT-AN94 mutagenic strain can achieve 70%; meanwhile mutagenic strain has good genetic stability, is conducive to later period amplification large-scale production；By the adjustment to fermentation medium nutritional ingredient, with mutagenic strain realize it is good compound, further promote the promotion of mutagenic strain YT-011A yield, there is preferable applicability；Especially gained YT-AN94 bacterial strain, in the case where optimizing fermentation medium, yield enhancing rate reaches 120%, has been obviously improved the economic benefit that YT-011A is produced by microbial fermentation engineering.
Description
Technical field
The present invention relates to technical field of bioengineering, and in particular to a kind of high yield YT-011A strain mutagenesis selection and
Its fermentation medium.
Background technique
It is on the rise the case where drug-fast bacteria infection at present, it is contemplated that the year two thousand fifty whole world or there are 10,000,000 people to die of drug-fast bacteria sense
Dye.Although the World Health Organization and national governments strengthen the reasonable employment to antibiotic by many kinds of measures, to slow down drug-fast bacteria
The further development of infection；But for catching with patient, just must not from drug-fast bacteria infection is solved the problems, such as at all
Disconnect the drug new varieties of hair antimicrobial agent.
Using active skull cap components as lead compound carry out structural modification be find antimicrobial agent new drug important channel and
Development trend, the glycopeptide class drug-resistance bacteria medicine that Present clinical uses mainly have vancomycin, teicoplanin, Dalbavancin and Austria
Sharp ten thousand magnitudes.Vancomycin and teicoplanin rise in the microbial severe infections of Gram-positive pathogens of confrontation multidrug resistant
Very important effect, and in recent years foreign countries listing Dalbavancin, oritavancin then be called treat these severe infections
" last line of defense " of property disease.
In Chinese patent CN107226845A, inventor is in teicoplanin, vancomycin, Dalbavancin, oritavancin etc.
On the basis of the pharmacology of glycopeptide class antibacterials, structure and technical study, by being improved to the acyl chain in Dalbavancin,
Have found the new glycopeptide class formation molecule YT-011 with very strong antibacterial activity.Drug efficacy study is demonstrate,proved in vitro the results show that YT-
011 coagulase-negative staphylococci (MRCNS) and staphylococcus aureus (MRSA) for methicillin resistance, benzyl penicillin
The enterococcus (VER) of drug resistant streptococcus pneumonia (PNSP) and drug resistance of vancomycin all has good bacteriostatic activity, and
Activity is better than Dalbavancin.Septicemia animal model is caused to carry out internal drug efficacy study using gram-positive bacterial infections ICR mouse
It has been shown that, YT-011 have good antibacterial action to gram positive bacteria, especially protect and make to drug-fast bacteria infection mouse septicemia model
With being substantially better than vancomycin.Therefore YT-011 has ideal development prospect.
As the synthesis precursor of YT-011 in the patent, YT-011A, i.e. Dalbavancin (Dalbavancin) precursor-
A40926, then from the village Ye Ye actinomyces (Nanomuria spp.) ATCC 39727.But currently, domestic and foreign literature is reported
The level of the village Ye Ye actinomyces (Nonomuriaspp.) fermenting and producing of Dalbavancin precursor producing strains is all lower, exists mostly
800mg/L or so.In order to improve the yield of A40926, domestic and foreign scholars have conducted extensive research work, mainly include that strain selects
It educates, two aspect of fermented and cultured optimization.
According to the literature, for many years, bacterium is produced by having carried out Dalbavancin precursor with a variety of different method of mutagenesis
The breeding of strain, obtains some superior strains.But method of mutagenesis is still in the majority with traditional physical mutagenesis and chemical mutagenesis, exists and lures
The limitations such as change method is random, direction is not easy to grasp, ideal individual screening difficulty.In recent years, there are some new means, such as
Cobalt ions, N~+ implantation are the synthesis method of mutagenesis for integrating physical mutagenesis and chemical mutagenesis, inhereditary material can be made to exist
It changes or lacks on gene level, significantly improve the aberration rate of biology, but this kind of equipment is expensive, it is not easy to operate, to make
It is restricted.
Summary of the invention
The present invention provides a kind of high yield YT-011A strain mutagenesis selection and its fermentation mediums, using ARTP-
NTG iteration complex mutation obtains the YT-011A high yield with genetic stability in conjunction with the breeding mode that streptomycin resistance screens
Bacterial strain, while also fermentation medium is optimized, to further increase producing strain, to solve existing Dalbavancin precursor
The method that production strain mutagenesis breeding and fermentation are cultivated promotes limited problem to producing strain.
The technical solution adopted by the invention is as follows:
A kind of high yield YT-011A strain mutagenesis selection, specifically: ARTP-NTG complex mutation is used to original strain
It is iterated mutagenesis, and is aided with streptomycin resistance breeding, obtains enhanced variant.
Further, the technical program meaning ARTP-NTG complex mutation refers to that ARTP mutagenesis and NTG alternately lure
Change is formed by comprehensive mutagenic processes.At the same time, ARTP mutagenesis and NTG mutagenesis are primary repeatedly per completion is alternately once calculated as
For mutagenesis, and preferred the number of iterations is 8 times in the technical program, that is, completes 8 groups of ARTP mutagenesis and NTG mutagenesis is constituted
ARTP-NTG complex mutation.In addition, every group of ARTP-NTG complex mutation, preferred NTG mutagenesis is carried out prior to ARTP mutagenesis.
Among the above, original strain refers to the bacterial strain with output YT-011A ability, specifically can be but be not limited to commercially available
In common 39727 bacterial strain of the village Ye Ye actinomyces ATCC.
ARTP mutagenesis, full name atmospheric pressure at room plasma (Atmospheric and Room Temperature
Plasma) induced-mutation technique, refer to using under atmospheric pressure generate temperature between 25~40 DEG C, have high activity particle (packet
Include helium atom, oxygen atom, nitrogen-atoms, 0H free radical etc. in excitation state) plasma jet of concentration, make microbial cell
Wall/
The structure and permeability changes of film, and cause gene damage, and then make microbial gene sequences and its metabolism network
Significant changes eventually lead to a kind of microorganism induced-mutation technique that microorganism generates mutation.
NTG mutagenesis, full name nitrosoguanidine (Nitroso-guanidin) induced-mutation technique refer to sub- using chemical supramutagen
Nitroguanidine makes microorganism generate a kind of microorganism induced-mutation technique being mutated.
Preferably, NTG mutagenesis is prior to ARTP mutagenesis every time in ARTP-NTG complex mutation.
Preferably, number of the original strain through iteration mutagenesis is 8 times.
In the technical program, using streptomysin to the centre in the original strain by mutagenic treatment or iteration mutagenic processes
Bacterial strain carries out resistant breeding, to improve the screening efficiency of direct mutation result during complex mutation.
Wherein, the method that bacterial strain carries out resistant breeding is obtained to by mutagenic treatment using streptomysin are as follows: the first is, each
Carrying out resistant breeding using streptomysin after mutagenesis will be by mutagenesis that is, after ARTP mutagenesis of every completion or NTG mutagenesis
Bacterial strain after reason, which is coated on streptomycin resistance plate, carries out breeding, then carries out NTG mutagenesis next time to the bacterial strain selected
Or ARTP mutagenesis, it so recycles, and be finally completed iteration and obtain enhanced variant.
Further, from original strain, per generation bacterial strain is through a wheel ARTP-NTG complex mutation and streptomycin resistance breeding
Afterwards, then by passage main inclined plane culture, after shake flask fermentation culture, the mutagenic strain with direct mutation is filtered out, for next
ARTP-NTG complex mutation and streptomycin resistance breeding are taken turns, is recycled with this, until completing iteration mutagenesis.Pass through passage main inclined plane training
It supports, the method for shake flask fermentation culture, realizes according to practical YT-011A yield, to direct mutation occurs in every generation mutagenic strain
Bacterial strain screening, reduce interference of the produced negative mutant strain to mutagenic and breeding in mutagenic processes, comprehensive entire iteration lures
From the point of view of change process, the screening efficiency of direct mutation result can be further increased.
Wherein, culture medium used in shake flask fermentation incubation is preliminary fermentation culture medium.The Preliminary fermentation training
The component of base is supported, by weight percentage, comprising: sodium chloride 0.1%, soluble starch 4.0%, cotton seed meal 1.0%, soybean powder
2.0%, yeast powder 1.0%, epsom salt 0.05%, glucose 2.0%, calcium carbonate 0.2%, residual mass is water；And its
PH value is 6.0~7.0.
Further, the condition of the ARTP mutagenesis is high-purity helium, power 120W, working gas flow 10L/min, mutagenesis
Distance 2mm, mutation time are 15~75s.
Further, the condition of the NTG mutagenesis is NTG concentration 2mg/L, handles 20~60min of time.
Further, the concentration of streptomysin used in the streptomycin resistance breeding is 0.2~0.8g/L.
By above-mentioned mutagenic breeding method, a kind of YT-011A yield can be obtained compared with the high yield that original strain significantly improves
Mutant strain.At the same time, a kind of fermentation medium of optimization has also been designed and provided in the technical program, it can be to bacterial strain
The yield of YT-011A is further promoted；It includes the following components: providing the carbon source of carbon for bacterial strain, the nitrogen of nitrogen is provided
Source provides the complex inorganic salt catalyst of inorganic salts substance, and provides the compound amino acid of amino acid substance；Wherein, the compound ammonia
Base acid is made of valine, isoleucine and sodium glutamate；In addition, above-mentioned carbon source, nitrogen source, complex inorganic salt catalyst and compound amino acid
It is uniformly dispersed by the auxiliary material allowed on bioengineering and forms corresponding fermentation medium, and the auxiliary material can be but not
It is limited to water.
Further, by the percentages for accounting for fermentation medium total weight, the carbon source contain 3.0~6.0% it is solvable
Property starch and 2.0~4.0% glucose；The nitrogen source contains 0.5~2.0% cotton seed meal, 2.0~4.0% soybean powder,
1.0~2.0% yeast powder；The complex inorganic salt catalyst contains 0.05~0.10% epsom salt, 0.1~0.5% chlorine
Change the calcium carbonate of sodium and 0.1~0.3%；The compound amino acid contains 0.1%~0.3% valine, 0.1~0.3%
Isoleucine, 0.5~1.5% sodium glutamate；In addition to the above components, residual mass is bioengineering in the fermentation medium
The auxiliary material of upper permission.
A kind of fermentation medium according to claim 9, it is characterised in that: bacterial strain is in the fermentation medium
The fermented and cultured period is 120~168h, and temperature is 32~34 DEG C, and pH value is 6.0~7.0.
In conclusion compared with the prior art, the invention has the advantages that:
(1) ARTP-NTG iteration complex mutation is used, is screened in conjunction with streptomycin resistance, the high yield YT-011A's of acquisition lures
Become bacterial strain, the yield increase rate of mutagenic strain generally reaches 50% or more, and wherein YT-AN94 mutagenic strain can achieve
70%；
(2) ARTP-NTG iteration complex mutation is used, is screened in conjunction with streptomycin resistance, the high yield YT-011A's of acquisition lures
Becoming bacterial strain has good genetic stability, is conducive to later period amplification large-scale production；
(3) by the adjustment to fermentation medium nutritional ingredient, with gained high yield mutagenic strain realize it is good compound,
The further promotion of mutagenic strain YT-011A yield is realized, when optimal set is used for AN94 bacterial strain, with initial incubation base phase
Than fermentation unit increase rate about 30%；
(4) YT-AN94 mutagenic strain can be obtained, in conjunction with optimization fermentation medium, ultimate output ability realize 1.6~
1.8g/L, compared with original strain and preliminary fermentation culture medium, enhancing rate reaches 120%, has been obviously improved micro-organisms YT-011A
The economic benefit of technique；
(5) mutagenic breeding method provided by the present invention is easy to operate, and mutation rate is high, and yield promotion effect is obvious, fits
In being widely used to promote.
Specific embodiment
All features disclosed in this specification can be with any other than mutually exclusive feature and/or step
Mode combines.
In order to make those skilled in the art more fully understand technical solution of the present invention, below with reference to specific embodiment
The present invention is described in further detail.
It should be noted that following Examples 1 to 9, since the application method of culture medium in mutagenesis and iteration mutagenesis belongs to
Common step in microorganism mutagenesis and incubation, those skilled in the art can select according to actual needs, therefore not
It is repeated in embodiments the step of.
Embodiment 1
A kind of high yield YT-011A strain mutagenesis selection, comprising the following steps:
To original strain by the mutagenesis choosing of " ARTP mutagenesis-streptomycin resistance screening-NTG mutagenesis-streptomycin resistance screening "
Sequence is educated, iteration mutagenesis 8 times, obtains enhanced variant.
Wherein, original strain refers to commercially available 39727 bacterial strain of the village Ye Ye actinomyces ATCC；
The condition of ARTP mutagenesis is high-purity helium, power 120W, working gas flow 10L/min, mutagenesis distance 2mm, mutagenesis
Time is 15s；
The condition of NTG mutagenesis is NTG concentration 2mg/L, handles time 20min；
The concentration of streptomysin used in streptomycin resistance breeding is 0.2g/L.
Embodiment 2
A kind of high yield YT-011A strain mutagenesis selection, comprising the following steps:
To original strain by the mutagenesis choosing of " ARTP mutagenesis-streptomycin resistance screening-NTG mutagenesis-streptomycin resistance screening "
Sequence is educated, iteration mutagenesis 8 times, obtains enhanced variant.
Wherein, original strain refers to commercially available 39727 bacterial strain of the village Ye Ye actinomyces ATCC；
The condition of ARTP mutagenesis is high-purity helium, power 120W, working gas flow 10L/min, mutagenesis distance 2mm, mutagenesis
Time is 75s；
The condition of NTG mutagenesis is NTG concentration 2mg/L, handles time 60min；
The concentration of streptomysin used in streptomycin resistance breeding is 0.8g/L.
Embodiment 3
A kind of high yield YT-011A strain mutagenesis selection, comprising the following steps:
To original strain by the mutagenesis choosing of " ARTP mutagenesis-streptomycin resistance screening-NTG mutagenesis-streptomycin resistance screening "
Sequence is educated, iteration mutagenesis 8 times, obtains enhanced variant.
Wherein, original strain refers to the commercially available village Ye Ye actinomyces ATCC39727 bacterial strain；
The condition of ARTP mutagenesis is high-purity helium, power 120W, working gas flow 10L/min, mutagenesis distance 2mm, mutagenesis
Time is 45s；
The condition of NTG mutagenesis is NTG concentration 2mg/L, handles time 40min；
The concentration of streptomysin used in streptomycin resistance breeding is 0.5g/L.
Embodiment 4
A kind of high yield YT-011A strain mutagenesis selection, comprising the following steps:
To original strain by the mutagenesis choosing of " NTG mutagenesis-streptomycin resistance screening-ARTP mutagenesis-streptomycin resistance screening "
Sequence is educated, iteration mutagenesis 8 times, obtains enhanced variant.
Wherein, original strain refers to commercially available 39727 bacterial strain of the village Ye Ye actinomyces ATCC；
The condition of ARTP mutagenesis is high-purity helium, power 120W, working gas flow 10L/min, mutagenesis distance 2mm, mutagenesis
Time is 15s；
The condition of NTG mutagenesis is NTG concentration 2mg/L, handles time 20min；
The concentration of streptomysin used in streptomycin resistance breeding is 0.2g/L.
Embodiment 5
A kind of high yield YT-011A strain mutagenesis selection, comprising the following steps:
To original strain by the mutagenesis choosing of " NTG mutagenesis-streptomycin resistance screening-ARTP mutagenesis-streptomycin resistance screening "
Sequence is educated, iteration mutagenesis 8 times, obtains enhanced variant.
Wherein, original strain refers to commercially available 39727 bacterial strain of the village Ye Ye actinomyces ATCC；
The condition of ARTP mutagenesis is high-purity helium, power 120W, working gas flow 10L/min, mutagenesis distance 2mm, mutagenesis
Time is 75s；
The condition of NTG mutagenesis is NTG concentration 2mg/L, handles time 60min；
The concentration of streptomysin used in streptomycin resistance breeding is 0.8g/L.
Embodiment 6
A kind of high yield YT-011A strain mutagenesis selection, comprising the following steps:
To original strain by the mutagenesis choosing of " NTG mutagenesis-streptomycin resistance screening-ARTP mutagenesis-streptomycin resistance screening "
Sequence is educated, iteration mutagenesis 8 times, obtains enhanced variant.
Wherein, original strain refers to commercially available 39727 bacterial strain of the village Ye Ye actinomyces ATCC；
The condition of ARTP mutagenesis is high-purity helium, power 120W, working gas flow 10L/min, mutagenesis distance 2mm, mutagenesis
Time is 45s；
The condition of NTG mutagenesis is NTG concentration 2mg/L, handles time 40min；
The concentration of streptomysin used in streptomycin resistance breeding is 0.5g/L.
Embodiment 7
The present embodiment provides a kind of fermentation medium that can be improved YT-011A yield, by accounting for fermentation medium total weight
Percentages, specifically:
The carbon source contains 4.0% soluble starch and 2.0% glucose；
The nitrogen source contains 0.5% cotton seed meal, 3.0% soybean powder, 1.0% yeast powder；
The complex inorganic salt catalyst contains 0.05% epsom salt, 0.3% sodium chloride and 0.1% calcium carbonate；
The compound amino acid contains 0.1% valine, 0.1% isoleucine, 0.5% sodium glutamate；
In addition to the above components, residual mass is the auxiliary material allowed on bioengineering in the fermentation medium.
Wherein, it is equal that carbon source, nitrogen source, complex inorganic salt catalyst and compound amino acid pass through the auxiliary material dispersion allowed on bioengineering
It is even and form corresponding fermentation medium, and the auxiliary material can be but be not limited to the water approved on bioengineering.
Wherein, fermented and cultured period of the enhanced variant in the fermentation medium is 120h, temperature 32
DEG C, fermentation pH value is 6.0.
Embodiment 8
The present embodiment provides a kind of fermentation medium that can be improved YT-011A yield, by accounting for fermentation medium total weight
Percentages, specifically:
The carbon source contains 6.0% soluble starch and 3.0% glucose；
The nitrogen source contains 1.0% cotton seed meal, 4.0% soybean powder, 1.0~2.0% yeast powder；
The complex inorganic salt catalyst contains 00.10% epsom salt, 0.5% sodium chloride and 0.3% calcium carbonate；
In addition to the above components, residual mass is the auxiliary material allowed on bioengineering in the fermentation medium.
Wherein, it is equal that carbon source, nitrogen source, complex inorganic salt catalyst and compound amino acid pass through the auxiliary material dispersion allowed on bioengineering
It is even and form corresponding fermentation medium, and the auxiliary material can be but be not limited to the water approved on bioengineering.
Wherein, fermented and cultured period of the enhanced variant in the fermentation medium be 168h, 34 DEG C of temperature,
PH value of fermenting is 7.0.
Embodiment 9
The present embodiment provides a kind of fermentation medium that can be improved YT-011A yield, by accounting for fermentation medium total weight
Percentages, specifically:
The carbon source contains 5.0% soluble starch and 2.5% glucose；
The nitrogen source contains 0.75% cotton seed meal, 3.5% soybean powder, 1.5% yeast powder；
The complex inorganic salt catalyst contains 0.075% epsom salt, 0.4% sodium chloride and 0.2% calcium carbonate；
In addition to the above components, residual mass is the auxiliary material allowed on bioengineering in the fermentation medium.
Wherein, it is equal that carbon source, nitrogen source, complex inorganic salt catalyst and compound amino acid pass through the auxiliary material dispersion allowed on bioengineering
It is even and form corresponding fermentation medium, and the auxiliary material can be but be not limited to the water approved on bioengineering.
Wherein, fermented and cultured period of the enhanced variant in the fermentation medium is 144h, temperature 33
DEG C, fermentation pH value is 6.5.
Test example
This test example is tested and is verified to technical solution of the present invention in terms of following two.
1, induction mutation of bacterium
Original strain through iteration complex mutation, and combines streptomycin resistance breeding under the conditions of ARTP-NTG complex mutation,
Secondary screening is verified by shaking flask, filters out that enhanced variant is several, and vacuum refrigeration is used to the enhanced variant filtered out
Pipe carries out preservation；Several verifyings have been carried out to mutant strain (mutagenic strain) to prove ARTP-NTG complex mutation of the present invention simultaneously
The validity of selection.
2, fermentation medium optimizes
By adding certain proportion valine, isoleucine, sodium glutamate into fermentation medium, through orthogonal test point
Analysis is optimized fermentation medium, and is cultivated using the fermentation medium enhanced variant, the fermentation training after peep optimization
It supports base and effect is promoted to the yield of enhanced variant.
Specific testing program is as follows:
One, experiment purpose
It is screened using ARTP-NTG iteration complex mutation combination streptomycin resistance, by shaking flask primary dcreening operation and secondary screening, selects height
Bacterial strain is produced, and verifies producing strain.
Two, experimental material
1, original strain and mutagenic strain
Original strain are as follows: 39727 bacterial strain of the village Ye Ye actinomyces ATCC.
Conceptual design: original strain is selected after NTG mutagenesis and ARTP mutagenic treatment according to positive mutation rate and lethality
The optimization process time；Then the spore suspension through NTG mutagenic treatment certain time is coated on anti-with certain density streptomysin
Cultivated on mild-natured plate, the single colonie grown on picking plate, production spore suspension body again through ARTP mutagenic treatment certain time,
Then it cultivates, the single colonie grown on picking plate, is passaged to tiltedly on certain density streptomycin resistance plate
After the growth and maturity of face, it is seeded to fermentation medium screening, the biggish several plants of bacterium of the direct mutation filtered out, preparation mixing spore hangs
Liquid repeats above complex mutation 8 times, completes iteration mutagenesis.Pay attention to carrying out the single colonie that mutagenesis screening comes out, is all made of " AN
The mode of XXX " is numbered, and wherein XXX indicates that the sequence screened according to single colonie artificial counting to be administered identifies, and
No longer change after given except this for the counting mark of the given mark bacterial strain, such as AN26 indicates to press selecting sequence the 26th
The single colonie after mutagenesis that position is screened out.
Best mutagenic condition: the condition of ARTP mutagenesis is high-purity helium, power 120W, working gas flow 10L/min, mutagenesis
Distance 2mm, mutation time are 15~75s；Wherein, mutation time can choose 15s, 20s, 25s, 30s, 35s, 40s, 45s,
50s、55s、60s、65s、70s、75s。
The condition of NTG mutagenesis is NTG concentration 2mg/L, handles 20~60min of time；Wherein, 20min, 25min, 30min,
35min、40min、45min、50min、55min、60min。
The concentration of streptomysin used in streptomycin resistance breeding be 0.2~0.8g/L, be specifically as follows 0.2g/L,
0.3g/L、0.4g/L、0.5g/L、0.6g/L、0.7g/L、0.8g/L。
It should be noted that above scheme mainly uses " NTG mutagenesis-streptomycin resistance screening-ARTP mutagenesis-streptomysin
The mutagenic and breeding sequence of resistance screening ", reduces the optimization of initial mutagenic and breeding condition and the choosing of enhanced variant
Educate the overall process sifted out；And pass through repetition test and find, other are such as " ARTP mutagenesis-streptomycin resistance screening-NTG mutagenesis-strepto-
The mutagenic and breeding sequence of plain resistance screening ", in the effect and variation tendency for obtaining mutagenesis and " NTG mutagenesis-streptomysin is anti-
Property screening-ARTP mutagenesis-streptomycin resistance screening " mutagenic and breeding sequence it is essentially identical, therefore, no longer expansion is said one by one herein
It is bright.
Among the above, for three NTG mutagenic treatment time, ARTP mutagenic treatment time and streptomysin concentration mutagenic and breedings
The most preferred range of controllable factor in the process can carry out parallel test for each factor by single-factor variable method, so respectively
Afterwards by handled under the conditions of single-factor variable bacterial strain positive mutation rate and lethality, close to judge and simultaneously determine each factor
Preferred scope and optimal value.Wherein, positive mutation rate is that the bacterial strain survived after being subject to processing yield occurs in shaking flask screening
The bacterial strain quantity of promotion accounts for the ratio of survival strains total quantity, for example, this unitary variant of time is handled for ARTP, through ARTP
After handling 60s, obtain 15 plants of bacterial strains, by initial medium shaking flask screening discovery yield have compared with starting strain the bacterium of promotion
Strain number amount is 7 plants, then positive mutation rate is 7/15 × 100%=46% under this condition.Lethality is parallel in above-mentioned single-factor variable
In test, then under the conditions of by NTG mutagenic treatment time, ARTP mutagenic treatment time or streptomysin concentration unitary variant
Reason, single colonie number is N on plate after processing, is M without single colonie number on any mutagenic treatment plate, then lethality is
2, seed culture medium: soluble starch 1.5%, glucose 1.0%, cotton seed meal 1.0%, soybean powder 0.5%, yeast
Powder 0.5%, pH6.0~7.0.
3, fermentation medium (hereinafter referred to as preliminary fermentation culture medium): sodium chloride 0.1%, soluble starch 4.0%, cotton seed meal
1.0%, soybean powder 2.0%, yeast powder 1.0%, epsom salt 0.05%, glucose 2.0%, calcium carbonate 0.2%, pH6.0
~7.0.
4, the fermentation medium (hereinafter referred to as fermentation medium or optimization fermentation medium) after optimizing: sodium chloride 0.1%, it can
Soluble starch 4.0%, cotton seed meal 1.0%, soybean powder 2.0%, yeast powder 1.0%, epsom salt 0.05%, glucose
2.0%, valine 0.1%~0.3%, isoleucine 0.1~0.3%, sodium glutamate 0.5~1.5%, calcium carbonate 0.2%,
PH6.0~7.0.
Three, laboratory operating procedures
1, it by prepared seed culture medium and fermentation medium, is dispensed respectively into 250mI triangular flask, liquid amount is
30mL, sterilize 30min at 120~122 DEG C, and cooling is spare.
2, it will be seeded in fermentation medium respectively through 8 iteration complex mutation obtained strains, in 32~34 DEG C, 220rpm
Lower culture 120h~168h carries out analysis detection to the fermentation liquid that culture obtains.
3, first time secondary screening: the superior strain obtained through primary dcreening operation is seeded in seed culture medium, 32~34 DEG C,
48h or so is cultivated under 220rpm, by cultured seed transferred species into fermentation medium, is cultivated at 32~34 DEG C, 220rpm
120h~168h carries out analysis detection to the fermentation liquid that culture obtains.This process is shaking flask screening, is screened by shaking flask, no
But further screening can be additionally aided with direct mutation to verifying the case where positive/negative mutation produced by mutagenic strain
Or the preferable mutagenic strain of direct mutation situation, to be further ensured that mutagenic breeding method is to bacterial strain direct mutation in the technical program
Screening efficiency.
4, fermentation medium Orthogonal Experiment and Design:
Starting strain: enhanced variant AN94, original strain
The design of culture medium reduced parameter:
4, second of secondary screening: the superior strain obtained through secondary screening and original strain are seeded in seed culture medium again,
48h or so is cultivated at 32~34 DEG C, 220rpm, by cultured bacterium seed transferred species to preliminary fermentation culture medium
(P10) it is cultivated with optimization fermentation medium (P1~P9), original strain seed liquor transferred species to preliminary fermentation culture medium (P10) kind,
120h~168h is cultivated at 32~34 DEG C, 220rpm, and analysis detection is carried out to the fermentation liquid that culture obtains.
5, fermentation liquid YT-011A content detection: add after taking fermentation liquid 3ml, NaOH tune pH to 10.0~11.0, concussion to mix
Enter 3mL ethyl alcohol, whirlpool shakes 5min, and 3000rpm is centrifuged 10min, takes supernatant to carry out high-efficient liquid phase analysis detection, according to area
Normalization method calculates YT-011A content.
Four, result
1 mutagenic strain shaking flask secondary screening result (first time secondary screening) of table
The results are shown in Table 1, passes through shaking flask in conjunction with streptomycin resistance plate screening through strain iteration multiple mutated
Screening obtains mutagenic strain AN94 than original strain output increased 69.53%；In addition to this, gained enhanced variant after mutagenesis
Yield enhancing rate generally 50% or more；Therefore, ARTP-NTG complex mutation breeding method provided by the present invention can have
Effect is promoted.
2 mutagenic strain AN94 shaking flask secondary screening result (second of secondary screening) of table
Note: increase rate refers to that bacterium is each compared with original strain is in preliminary fermentation culture medium fermentability in upper table
Fermentability increase rate in Optimal Medium and preliminary fermentation culture medium.
The results are shown in Table 2, and original strain is handled through iteration complex mutation (ARTP-NTG), in conjunction with streptomycin resistance plate
Screening, screens to obtain mutagenic strain AN94 than original strain output increased 71% by shaking flask, demonstrates gained in a secondary screening
The yield increase rate of AN94.For optimization fermentation medium, ratio is compounded according to each aminoacid ingredient content in compound helium base acid
The difference of example, compared with initial medium, identical strain fermentation unit has promotion, especially AN94 bacterial strain to ferment in optimal set
Culture medium fermentation unit increase rate is up to 30%.
It according to result above, selects optimal set for P8, i.e., adds valine 0.3%, isoleucine in fermentation medium
0.2%, sodium glutamate 0.5%, the superior strain YT-AN94 obtained through iteration Mutation screening is on optimization fermentation medium
Fermentation unit compared with fermentation unit, improves in preliminary fermentation culture medium up to 1.8g/L or so with original starting strain
119%.
In addition, test in, each Optimal Medium to other mutagenic strains rise raising output results the regularity of distribution with lure
It is identical to become the rule that strains A N94 is reflected, therefore is no longer enumerated here.
To sum up, it can be proved that optimization fermentation medium provided in the present invention, by the selection of amino acid classes and multiple
Adjusting with ratio realizes the further promotion of enhanced variant YT-011A yield.
Embodiment 2:
This test example carries out investigation verifying, thinking mainly for the mitotic stability of enhanced variant are as follows: to screening
Several enhanced variants out carry out continuous inclined-plane passage, and continuous passage 5 times, shaking flask verifying is each on optimization fermentation medium
Enhanced variant fermentation produces the genetic stability of YT-011A ability.
Mitotic stability test is specific as follows:
One, experiment purpose
Obtained superior strain is subjected to mitotic stability test, determines whether mutant strain has good inheritance stability
Property.
Two, experimental material
1, mutagenic strain YT-AN94.
2, seed culture medium: soluble starch 1.5%, glucose 1.0%, cotton seed meal 1.0%, soybean powder 0.5%, yeast
Powder 0.5%, pH6.0~7.0.
4, the fermentation medium after optimizing: sodium chloride 0.1%, soluble starch 4.0%, cotton seed meal 1.0%, soybean powder
2.0%, yeast powder 1.0%, epsom salt 0.05%, glucose 2.0%, valine 0.3%, isoleucine 0.2%, paddy ammonia
Sour sodium 0.5%, calcium carbonate 0.2%, pH6.0~7.0.
Three, laboratory operating procedures
1, it by prepared slant medium, dispenses into test tube, sterilize 30min at 120~122 DEG C, spare after cooling.
2,48~72h of blank culture will be carried out at inclined-plane after cooling in the incubator 37 DEG C.
3, mutagenic strain YT-AN94 is inoculated on inclined-plane, carries out secondary culture, inclined-plane is placed in 28~30 DEG C of incubators
Culture 7 days or so, cultured bacterial strain is placed in 4 DEG C of refrigerators and saves, for use.This process is to pass on main inclined plane culture.
4, it by cultured inclined-plane, is inoculated in seed culture medium by digging block method, is cultivated at 32~34 DEG C, 220rpm
48h or so, by cultured seed transferred species to optimization post-fermentation culture medium in, at 32~34 DEG C, 220rpm cultivate 120~
168h carries out analysis detection to the fermentation liquid that culture obtains.
5, fermentation liquid YT-011A content detection: after taking fermentation liquid 3ml, NaOH tune pH10.0~11.0 or so, concussion to mix
3ml ethyl alcohol is added, whirlpool shakes 5min, and 3000rpm is centrifuged 10min, takes supernatant to carry out high-efficient liquid phase analysis detection, according to face
Product normalization method calculates YT-011A content.
Four, result
The mitotic stability test result of 1 mutagenicity high-yield bacterial strain YT-AN94 of table
As it can be seen from table 1 mutagenic strain YT-2088-AN94 has preferable genetic stability within 5 generations.
Conclusion: multiple using novel ARTP-NTG iteration using the village starting strain Ye Ye Pseudomonas ATCC39727 as starting strain
Mutagenesis is closed, in conjunction with streptomycin resistance breeding, a plant height is obtained and produces the superior strain YT- that YT-011A yield is 1.2~1.4g/L
AN94, and the bacterial strain inheritance stability show that ARTP-NTG compound mutation breeding is the one of effective and feasible raising YT-011A yield
Kind mutation breeding means.Meanwhile fermentation medium is optimized, it is trained with YT-AN94 and original starting strain in Preliminary fermentation
Base phase ratio is supported, yield is respectively increased 50%, 120% to bacterial strain YT-AN94 on fermentation medium after optimization, it can be seen that, pass through
Optimization fermentation medium can also effectively improve YT-011A yield.
The specific embodiment of the application above described embodiment only expresses, the description thereof is more specific and detailed, but simultaneously
The limitation to the application protection scope therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, under the premise of not departing from technical scheme design, various modifications and improvements can be made, these belong to this
The protection scope of application.
Claims (10)
1. a kind of high yield YT-011A strain mutagenesis selection, which is characterized in that the mutagenic breeding method are as follows: to original bacteria
Strain is iterated mutagenesis using ARTP-NTG complex mutation, and is aided with streptomycin resistance breeding, obtains enhanced variant.
2. a kind of YT-011A strain mutagenesis selection according to claim 1, it is characterised in that: the ARTP-NTG
Complex mutation is ARTP mutagenesis and NTG mutagenesis alternately；The streptomycin resistance breeding is will be through the bacterium after each mutagenesis
Strain, which is coated in streptomycete resistant panel, carries out breeding.
3. a kind of YT-011A strain mutagenesis selection according to claim 2, it is characterised in that: from original strain,
Per generation bacterial strain is after a wheel ARTP-NTG complex mutation and streptomycin resistance breeding, then passes through passage main inclined plane culture, shaking flask hair
Ferment culture carries out secondary screening, and it is anti-for next round ARTP-NTG complex mutation and streptomysin to filter out the mutagenic strain with direct mutation
Property breeding, recycled with this, until complete iteration mutagenesis.
4. described in any item a kind of high yield YT-011A strain mutagenesis selections according to claim 1~3, feature
In: the condition of the ARTP mutagenesis be high-purity helium, power 120W, working gas flow 10L/min, mutagenesis distance 2mm, when mutagenesis
Between be 15~75s.
5. described in any item a kind of high yield YT-011A strain mutagenesis selections according to claim 1~3, feature
In: the condition of the NTG mutagenesis is NTG concentration 2mg/L, handles 20~60min of time.
6. described in any item a kind of high yield YT-011A strain mutagenesis selections according to claim 1~3, it is characterised in that:
Number of the original strain through iteration mutagenesis is 8 times.
7. a kind of high yield YT-011A strain mutagenesis selection according to claims 1 to 3, it is characterised in that: the chain
The concentration of streptomysin used in chloramphenicol resistance breeding is 0.2~0.8g/L.
8. a kind of fermentation medium for any one of claim 1~7 mutagenic breeding method, which is characterized in that including
Following components: the auxiliary material allowed on carbon source, nitrogen source, complex inorganic salt catalyst, compound amino acid and bioengineering；The compound ammonia
Base acid is made of valine, isoleucine and sodium glutamate.
9. a kind of fermentation medium according to claim 8, by the percentages for accounting for fermentation medium total weight, feature
Be: the carbon source contains 4.0~6.0% soluble starch and 2.0~3.0% glucose；The nitrogen source contains 0.5~
1.0% cotton seed meal, 3.0~4.0% soybean powder, 1.0~2.0% yeast powder；The complex inorganic salt catalyst contains 0.05~
0.10% epsom salt, 0.3~0.5% sodium chloride and 0.1~0.3% calcium carbonate；The compound amino acid contains
0.1%~0.3% valine, 0.1~0.3% isoleucine, 0.5~1.5% sodium glutamate；In addition to the above components,
Residual mass is the auxiliary material allowed on bioengineering in the fermentation medium.
10. a kind of fermentation medium according to claim 9, it is characterised in that: bacterial strain is in the fermentation medium
The fermented and cultured period is 120~168h, and fermented and cultured temperature is 32~34 DEG C, and fermented and cultured pH value is 6.0~7.0.
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