CN109762757A - A kind of high yield Co-Q10 hydrogenlike silicon ion and its mutagenic and breeding and application - Google Patents
A kind of high yield Co-Q10 hydrogenlike silicon ion and its mutagenic and breeding and application Download PDFInfo
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Abstract
The present invention provides a kind of high yield Co-Q10 hydrogenlike silicon ion and its mutagenic and breeding and applications, are related to microorganism mutation breeding field.It is bacterium germination with the bacterial strain by space mutagenesis screening, atmospheric pressure at room plasma mutagenesis has been carried out to it, through multiple resistance screening, high flux screening and amplification verifying screening step by step, a kind of high yield Co-Q10 hydrogenlike silicon ion is obtained, it is preserved in China General Microbiological culture presevation administrative center, deposit number is CGMCC No.16625.The Rhodobacter sphaeroides mutant strain that the present invention obtains, through 20m3Fermenting and producing verifying, mean titre are significantly improved up to 2694 μ g/mL, the yield of Co-Q10, in addition, the bacterial strain also has quadruple resistance, are had a extensive future.
Description
Technical field
The present invention relates to microorganism mutation breeding fields, and in particular to a kind of high yield Co-Q10 hydrogenlike silicon ion and its lures
Become breeding and application.
Background technique
Co-Q10 (CoenzymeQ10, CoQ10) is also known as ubiquinone, chemical name 2, the 3- dimethoxy -5- methyl -6- last of the ten Heavenly stems
Iso-amylene benzoquinones is a kind of biostearin substance, is present in animal, plant, microorganism etc. into the cell with extremely low content, and energy
It is synthesized in all organism tissues.Co-Q10 is the required coenzyme and natural antioxidant for generating ATP, participates in cell
Metabolic activity can activate improvement human body cell, enhance human immune system's function, be widely used to health care product, food, change
The industries such as cosmetic.
The method of production Co-Q10 raw material mainly has biological extraction method, chemical synthesis and microbe fermentation method, wherein micro-
Biological fermentation process bioactivity is high, cost of material is low and can amplify raising production capacity by scale, is acknowledged as most excellent
A kind of technical matters of gesture and potentiality.The bacterial strain for producing Co-Q10 in nature mainly has Rhodospirillum (Rhodospirillum
Molisch), Agrobacterium (Agrobacterium Conn), paracoccus (Paracoccus Davis), pseudomonas
(Pseudomonasaeruginosa), candida (Candida) etc., wherein hydrogenlike silicon ion (Rhodobacter
It sphaeroides is) at present for one of the main production bacterial strain of large scale fermentation production Co-Q10.Hydrogenlike silicon ion is logical
While cross cyclic photophosphorylation process and obtain growth and breeding energy, the Co-Q10 that needs largely to accumulate cuts down photosynthetic work
With the free radical of generation.The accumulation of usual wild-type microorganisms Co-Q10 only has (0.8-3.3) mg/g DCW, fermentation level
(30-130) mg/L, is unable to satisfy the requirement of industrial-scale fermenting and producing.Currently, generally by high-yield strains breeding with
The microbial fermentation that transformation and fermentation condition and two kinds of approach of optimization of process control improve Co-Q10 is horizontal, obtains Co-Q10
For the main method of superior strain still using traditional either physically or chemically carry out mutation breeding, physical mutagenesis has method letter
The advantages that single, highly-safe, at low cost and harvest is big.
Atmospheric pressure at room plasma (ARTP) is referred to as the 4th state of the substance in addition to gas, liquid, solid, passes through change
Mode of excitation and generator architecture can produce the plasma of different thermodynamic states.Plasma has ozone concentration and purple
The features such as external radiation intensity is extremely low, highly-safe, environmental-friendly, mutagenesis is quick, atmospheric pressure at room plasma mutagenesis operation letter
Single, mild condition, bacterial strain mutation rate is high, be mutated point and span is extensive.ARTP work gas source category, flow, discharge power, place
It is controllable to manage the conditions such as time, by changing instrumentation condition, the intensity and mutation storage capacity of strain mutation can be greatly improved
Amount, combination pressure screening and High Throughput Screening Assay, ARTP have become the new method of efficient evolution breeding.ARTP mutagenesis is general
Using lethality as the index of Screening, Mutation condition etc., lethality is unsuitable too high or too low, and some researches show that lethality is closer
90%, Mutagenic Effect is better, and mutagenic condition at this time is better.Currently, Chinese patent, which announces CN105505915A, discloses one kind
Using the method for ARTP mutagenesis screening high activity tolerance formaldehyde degrading bacteria mutant strain, mutagenic condition is power 115W, work
Throughput 10L/min, the distance between plasma emission source and bacterial strain 2mm, 23.0-35.0 DEG C of operation temperature, mutagenic treatment
Time 0.5-3min.
Space Mutation Breeding was widely used in Microbial Breeding in recent years, and achieved certain achievement, and party is of heap of stone etc.
(2010) a kind of method using space mutagenesis breeding Co-Q10 Producing Strain is disclosed, it is red to class ball using space carrying technology
Bacterium carries out space mutagenesis breeding, finally obtains the mutant strain of entitled Shenzhou6, yield is up to (0.8 ± 0.02) g/L.Though
Right class Rhodococcus sp carries out space mutagenesis, strain performance, which has, to be substantially improved through multiple space carrying.But due to same mutagenesis method
Mainly cause the variation in the fixed site of the certain genes of strain, bacterial strain has had certain resistance, breeding bacterium after repeatedly carrying
Kind performance boost is unobvious, occurs " fatigue phenomenon ", constrains further increasing for Co-Q10 strain fermentation level.Therefore,
Mutagenesis again is carried out to it using a kind of novel mutagenesis method on the basis of space carrying mutation breeding, increases Mutagenesis Site,
Screening high-performance mutant strain is very important.
Summary of the invention
The purpose of the present invention is to provide a kind of high yield Co-Q10 hydrogenlike silicon ion and its mutagenic and breeding and applications.In sky
Between ARTP mutagenesis is carried out on the basis of mutation breeding, overcome the problems of the prior art, improve the yield of Co-Q10.
One aspect of the present invention provides a kind of high yield Co-Q10 hydrogenlike silicon ion, strain number CPF, has been preserved in
State's General Microbiological Culture preservation administrative center, deposit number are CGMCC No.16625.
Another aspect of the present invention provides a kind of mutagenic breeding method of high yield Co-Q10 hydrogenlike silicon ion, the side
Method the following steps are included:
(1) preparation of seed bacteria suspension: activation culture hydrogenlike silicon ion starting strain CF02, picking single colonie, 32 DEG C,
220rpm is cultivated for 24 hours, obtains seed bacteria suspension；
The deposit number of the hydrogenlike silicon ion starting strain CF02 is CGMCC NO.2458, is by space mutagenesis
What screening obtained；
(2) preparation of bacteria suspension slide glass: taking seed bacteria suspension, and supernatant is removed in centrifugation, the physiology salt for being 0.8% with mass ratio
After bacterial sediment is resuspended in water, with diluted to OD600=0.6-0.8, obtains bacteria suspension, and bacteria suspension is uniformly applied to metal
ARTP mutagenesis samples are made on slide glass；
The physiological saline that the dilution is the glycerol that volume ratio is 5% and mass ratio is 0.8% according to 1:5 volume
Than mixing；
(3) ARTP mutagenesis: using ARTP-II type instrument to ARTP mutagenesis samples carry out mutagenesis, using 99.99% helium as
Working gas, power 100-120W, mutagenesis distance 1-3mm, mutation time 20-30s, protective agent is that concentration expressed in percentage by volume is
The glycerol of 5%-15%, after mutagenesis, vortex oscillation 1-3min elutes bacterium solution, the bacteria suspension after obtaining mutagenesis；
Preferably, power described in step (3) is 120W, mutagenesis distance is 2mm, mutation time 20s, protection
Agent is the glycerol that concentration expressed in percentage by volume is 5%.
(4) screening of bacterial strain: multiple resistance screening, high flux screening are carried out to the bacteria suspension after mutagenesis and step by step amplified
Verifying screening, obtains the high yield Co-Q10 hydrogenlike silicon ion.
Specifically, the preparation concrete operations of seed bacteria suspension described in step (1) are as follows:
The activation culture hydrogenlike silicon ion starting strain CF02 in culture medium A, 32 DEG C of culture 5d；Picking single colonie, inoculation
In the seed bottle containing culture medium B, 32 DEG C, 220rpm is cultivated for 24 hours, obtains seed bacteria suspension；
The culture medium A includes: yeast powder 1.0-10.0g/L, potassium dihydrogen phosphate 0.1-2.5g/L, magnesium sulfate 1.0-
5.0g/L, ferrous sulfate 0.1-1.0g/L, sodium chloride 0-5.0g/L, ammonium sulfate 1.5-3.5g/L, sodium glutamate 0.5-3.0g/L,
Dried Corn Steep Liquor Powder 0.5-3.0g/L, glucose 5-20g/L, agar 15-20g/L, pH=6.5-7.3；
The culture medium B includes: yeast powder 1.0-10.0g/L, potassium dihydrogen phosphate 0.1-2.5g/L, magnesium sulfate 1.0-
5.0g/L, ferrous sulfate 0.1-1.0g/L, sodium chloride 0-5.0g/L, ammonium sulfate 1.5-3.5g/L, sodium glutamate 0.5-3.0g/L,
Dried Corn Steep Liquor Powder 0.5-3.0g/L, glucose 5-20g/L, pH=6.5-7.3.
Preferably, the culture medium A includes: yeast powder 5.0g/L, potassium dihydrogen phosphate 0.5g/L, potassium dihydrogen phosphate
0.5g/L, epsom salt 1.5g/L, ferrous sulfate heptahydrate 0.1g/L, sodium chloride 2.5g/L, ammonium sulfate 2.5g/L, sodium glutamate
1.0g/L, Dried Corn Steep Liquor Powder 2.5g/L, glucose 10g/L, agar 15-20g/L, pH=7.16；
The culture medium B includes: yeast powder 5.0g/L, potassium dihydrogen phosphate 0.5g/L, potassium dihydrogen phosphate 0.5g/L, seven water
Magnesium sulfate 1.5g/L, ferrous sulfate heptahydrate 0.1g/L, sodium chloride 2.5g/L, ammonium sulfate 2.5g/L, sodium glutamate 1.0g/L, corn
Paste dry powder 2.5g/L, glucose 10g/L, pH=7.16.
Specifically, the screening of multiple resistance described in step (4) includes the following steps:
Bacteria suspension rejuvenation after the mutagenesis for taking step (3) to obtain, is inoculated in screening and culturing medium 1 and cultivates, and obtains anti-coenzyme
The substance resistant strain of Q10 analogue is referred to as R1；
ARTP mutagenesis is carried out by starting strain of R1, rejuvenation is inoculated in screening and culturing medium 2 and cultivates, in R1 underlying resistance
On obtain the double resistant strains of anti-respiratory chain ubiquinone inhibitor and be referred to as R2；
ARTP mutagenesis is carried out by starting strain of R2, rejuvenation is inoculated in screening and culturing medium 3 and cultivates, in R2 underlying resistance
On obtain triple resistant strains of anti-cell toxicant and be referred to as R3；
ARTP mutagenesis is carried out by starting strain of R3, rejuvenation is inoculated in screening and culturing medium 4 and cultivates, in R3 underlying resistance
On obtain the quadruple resistant strain of anti-Co-Q10 precursor analog and be referred to as R4；
The screening and culturing medium 1 is that joined Co-Q10 analogue in culture medium A；
The screening and culturing medium 2 is that joined respiratory chain ubiquinone inhibitor in culture medium A；
The screening and culturing medium 3 is that joined cytotoxic substance in culture medium A；
The screening and culturing medium 4 is that joined Co-Q10 precursor analog in culture medium A；
The screening and culturing: every kind of resisting substance repeats screening 3 times, 32 DEG C of culture 5d；
Bacterial strain rejuvenation method after the mutagenesis are as follows: take the bacteria suspension obtained after 1mL mutagenesis in culture medium B, 32 DEG C,
220rpm recovery 2h after being diluted to OD600=0.6-0.8, takes 100 μ l dilutions to be coated with resistant panel, flat for subsequent resistance
Screen choosing.More excellent bacterial strain after screening is subjected to preservation, constructs bacterial strain mutation library.
The bacterial strain method for preserving are as follows: the single bacterium selected is fallen in 50mL/250mL seed liquid culture medium, and 32 DEG C,
220rpm culture obtains seed liquor for 24 hours, by seed liquor: 50% glycerol=1:1 volume ratio, and it is low to deposit in -80 DEG C of refrigerators progress
Temperature saves.
Preferably, the Co-Q10 analogue is 5mg/L Vitamin K3 or 55.6mg/L adriamycin；
The respiratory chain ubiquinone inhibitor is 1.25mg/L Na3N or 566mg/L Na2S；
The cytotoxic substance is 3.25mg/L actinomycin D；
The Co-Q10 precursor analog is 250mg/L benzoic acid or 150mg/L P-hydroxybenzoic acid.
Specifically, specific step is as follows for high flux screening described in step (4):
Take above-mentioned R4 in the deep-well plates containing culture medium B, 220rmp, 32 DEG C of cultures obtain seed liquor for 24 hours；By seed
Liquid, which is inoculated in culture medium C, carries out fermented and cultured, and inoculum concentration 2%, dress liquid coefficient be 70%, shaking speed 220rpm, and 32
DEG C culture 72h, liquid phase detect fermentation liquid potency, screening Co-Q10 relative productivity 6.4% or more bacterial strain as high-throughput sieve
Select bacterium；
The culture medium C includes: Dried Corn Steep Liquor Powder 2.0g/L, sodium glutamate 2.5g/L, ammonium sulfate 3.0g/L, vulcanized sodium
1.5g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 5.0g/L, calcium carbonate 5.0g/L, glucose 20g/L, pH=7.10-7.20；
The liquid phase testing conditions are as follows: wavelength=275nm, the 10 reversed column of μ l, 4.6mm*150mm C18 of sample volume, column
32 DEG C, flow velocity 1mL/min, runing time 20min of temperature.
Specifically, the deep-well plates fermentation liquid titer detection method: cultured mutant strain fermentation liquid is centrifuged through orifice plate
Machine, 4500rmp are centrifuged 10min, remove the water phase in fermentation liquid, 300 μ l supernatants are taken, through (0.45 μm of vacuum hole panel filter
Filter membrane) it filters three times, it is final to obtain 1mL filtrate, the centrifuge tube of sterilizing-drying is moved it into, 30% hydrogen peroxide is added into pipe
0.1mL and 0.1M hydrochloric acid 0.1mL is settled to 10mL with ethyl alcohol, and when imbibition, the wall built-up mycelia of flushing hole inner wall is blown and beaten repeatedly, mixes
Even fermentation liquid when liquid relief, is blown and beaten repeatedly, wall built-up fermentation liquid in pipette tips is gone in centrifuge tube, after being mixed by inversion, is put into ultrasonic wave
In washer, ultrasonic 1h takes out centrifuge tube from ultrasonic cleaner, is mixed by inversion, and stands 15min, takes upper layer sample liquid 300
μ l is added in 96 hole filters, and filtrate is filtered through 0.45 μm of organic phase filter membrane, draws filtrate 1mL in liquid-phase inlet bottle,
It is detected with liquid phase.
Specifically, amplification verifying screening step includes: the high pass for taking claim 5 to obtain step by step described in step (4)
Amount screening bacterium is screened through shaking flask, and 2L tetrad tank fermentation, first class seed pot culture, secondary seed tank culture transfers to 20m3Hair
Fermented and cultured in fermentation tank, screening obtain high yield Co-Q10 hydrogenlike silicon ion.
More specifically, amplification verifying screening step includes: step by step described in step (4)
Shaking flask screening: taking high flux screening bacterium through culture medium A plate culture, culture medium B seed culture, inoculates containing to having
In the shaking flask of culture medium C, 32 DEG C, 220rpm is cultivated 3 days, is carried out liquid phase detection, is filtered out fermentation titer more than 528 μ g/mL
Bacterial strain, be referred to as strains A；
The fermentation of 2L tetrad tank: the strains A that shaking flask is screened is through culture medium A plate culture, culture medium B seed culture,
It inoculates in the 2L tetrad tank containing culture medium C, 32 DEG C of culture 90h, during which supplements feed supplement 1 three times, carry out liquid phase detection, sieve
Bacterial strain of the fermentation titer more than 2010 μ g/mL is selected, bacterial strain B is referred to as；
20m3Fermentor screening: the bacterial strain B that 2L tetrad tank is fermented is through culture medium A plate culture, culture medium B seed
It cultivates, is cultivated for 24 hours in the first class seed pot of culture medium D, cultivate 16h in the secondary seed tank of culture medium E, transfer to containing training
Support the 20m of base F3During which fermentation cylinder for fermentation culture supplements feed supplement 2 three times, cultivate 90h, carries out liquid phase detection, and screening obtains height
Produce Co-Q10 hydrogenlike silicon ion.
Specifically, the culture medium D includes: glucose 4g/L, ammonium sulfate 2.5g/L, dipotassium hydrogen phosphate 0.5g/L, phosphorus
Acid dihydride potassium 0.5g/L, magnesium sulfate 2g/L, ferrous sulfate 1g/L, calcium carbonate 35g/L, defoaming agent 0.3g/L, Dried Corn Steep Liquor Powder
0.5g/L, sodium glutamate 0.5g/L, manganese sulfate 0.03g/L, cobalt chloride 0.001g/L, yeast powder 1.0g/L, pH=7.10-
7.20；
The culture medium E includes: glucose 12.6g/L, ammonium sulfate 2.5g/L, dipotassium hydrogen phosphate 0.63g/L, di(2-ethylhexyl)phosphate
Hydrogen potassium 0.63g/L, magnesium sulfate 2.9g/L, ferrous sulfate 0.15g/L, defoaming agent 0.17g/L, Dried Corn Steep Liquor Powder 0.84g/L, paddy ammonia
Sour sodium 0.63g/L, manganese sulfate 0.04g/L, cobalt chloride 0.013g/L, yeast powder 2.1g/L, pH=7.10-7.20；
The culture medium F includes: ammonium sulfate 3.4g/L, potassium dihydrogen phosphate 0.4g/L, magnesium sulfate 10.4g/L, and sulfuric acid is sub-
Iron 1.12g/L, sodium chloride 2.1g/L, defoaming agent 0.13g/L, calcium chloride dihydrate 0.06g/L, corn pulp 7.5g/L, sodium glutamate
2.4g/L, manganese sulfate 0.05g/L, pH=7.10-7.20；
Material 1 in the feed supplement 1 includes: Dried Corn Steep Liquor Powder 6.21g/L, sodium glutamate 1.55g/L, ammonium sulfate 1.55g/
L, potassium dihydrogen phosphate 0.62g/L, magnesium sulfate 5.43g/L, ferrous sulfate 0.47g/L, sodium chloride 1.24g/L, manganese sulfate 0.02g/
L, calcium chloride 0.03g/L, defoaming agent 0.15g/L, pH=7.10-7.20；
Material 2 in the feed supplement 2 includes: ammonium sulfate 5.3g/L, potassium dihydrogen phosphate 2g/L, magnesium sulfate 23g/L, and sulfuric acid is sub-
Iron 2.1g/L, sodium chloride 5.1g/L, defoaming agent 1g/L, calcium chloride dihydrate 0.12g/L, Dried Corn Steep Liquor Powder 1.0g/L, sodium glutamate
6.0g/L, manganese sulfate 0.12g/L, pH=7.10-7.20.
The sterilising conditions of the above culture medium are 121 DEG C of sterilizing 30min；Mend sugared sterilising conditions: 60% liquid sugar uses 115.5
± 0.5 DEG C of sterilizing 25min；Liquid phosphorus sterilising conditions: 121 ± 1 DEG C of sterilizing 30min of 0.08g/L potassium dihydrogen phosphate.
The present invention also provides a kind of application of high yield Co-Q10 hydrogenlike silicon ion in fermentation preparation of cozymase Q 10；
Compared with prior art, positive and beneficial effect of the invention is:
1, the present invention has carried out atmospheric pressure at room plasma (ARTP) to it based on the bacterial strain by space mutagenesis screening
Mutagenesis has obtained a kind of high yield Co-Q10 class ball through multiple resistance screening, high flux screening and amplification verifying screening step by step
Red bacterium, has been preserved in China General Microbiological culture presevation administrative center, and deposit number is CGMCC No.16625.The bacterium
Strain, through 20m3Fermenting and producing verifying, mean titre are improved up to 2694 μ g/mL, the fermentation level for the bacteria strain CF02 that sets out
9.8%, the yield of Co-Q10 significantly improves, in addition, the bacterial strain also has quadruple resistance, has broad application prospects.
2, mutagenesis screening method provided by the invention optimizes the condition of ARTP mutagenesis, is more suitable for luring for hydrogenlike silicon ion
Become, has expanded ARTP mutation breeding range；The screening for increasing multiple resistance bacterial strain, makes bacterial strain obtain multiple resistance；Using
The devices such as 24 hole deep-well plates, orifice plate centrifugation and evacuated panel filtering, significantly reduce material screening cost, improve working efficiency and
Screen flux；Through shaking flask, 2L tetrad tank, 20m3Fermentor amplifies verifying step by step and carries out secondary screening, comprehensive and systematic completion to bacterial strain
The screening of bacterial strain, obtained bacterial strain genetic stability is good, with having important economic value and social effect.
Detailed description of the invention
Fig. 1 is high flux screening result figure in embodiment 4；
Fig. 2 is the fermentation titer histogram of shake flask fermentation in embodiment 5；
Fig. 3 is 2L tetrad tank and 20m in embodiment 53The fermentation titer histogram of fermentor；
Fig. 4 is mutant strain CPF and starting strain CF02 in embodiment 5 in 20m3The potency pair of fermentation cylinder for fermentation process
Than figure.
Specific embodiment
The explanation of following embodiment is merely used to help understand method and its core concept of the invention.It should be pointed out that pair
For those skilled in the art, without departing from the principle of the present invention, the present invention can also be carried out
Some improvements and modifications, these improvements and modifications also fall within the scope of protection of the claims of the present invention.To disclosed implementation
The following the description of example, enables those skilled in the art to implement or use the present invention.Various modifications to these embodiments
It will be readily apparent to those skilled in the art, the general principles defined herein can not depart from this
In the case where the spirit or scope of invention, realize in other embodiments.Therefore, the present invention is not intended to be limited to illustrated herein
These embodiments in, but can be applied to meet broader model consistent with the principles and novel features disclosed in this article
It encloses.Although can be used in implementation or test of the invention and heretofore described similar or of equal value any method and material
Material, preferred method and material are enumerated by place herein.
Unless otherwise defined, all technical and scientific terms used herein have and the technical field of the invention
The normally understood identical meaning of those of ordinary skill.
In the examples where no specific technique or condition is specified, described technology or conditions according to the literature in the art
(such as with reference to J. Pehanorm Brooker etc. write, " Molecular Cloning:A Laboratory guide " that Huang Peitang etc. is translated, the third edition, Science Press) or
Person carries out according to product description.
The culture medium prescription used in specific embodiment is as follows:
Culture medium A: yeast powder 5.0g/L, potassium dihydrogen phosphate 0.5g/L, potassium dihydrogen phosphate 0.5g/L, epsom salt
1.5g/L, ferrous sulfate heptahydrate 0.1g/L, sodium chloride 2.5g/L, ammonium sulfate 2.5g/L, sodium glutamate 1.0g/L, Dried Corn Steep Liquor Powder
2.5g/L, glucose 10g/L, agar 15-20g/L, pH=7.16.
Culture medium B: not adding agar, other are consistent with plating medium formula.
Culture medium C: Dried Corn Steep Liquor Powder 2.0g/L, sodium glutamate 2.5g/L, ammonium sulfate 3.0g/L, vulcanized sodium 1.5g/L, phosphorus
Acid dihydride potassium 0.5g/L, magnesium sulfate 5.0g/L, calcium carbonate 5.0g/L, glucose 20g/L, pH=7.10-7.20.
Material 1 in feed supplement 1: Dried Corn Steep Liquor Powder 6.21g/L, sodium glutamate 1.55g/L, ammonium sulfate 1.55g/L, biphosphate
Potassium 0.62g/L, magnesium sulfate 5.43g/L, ferrous sulfate 0.47g/L, sodium chloride 1.24g/L, manganese sulfate 0.02g/L, calcium chloride
0.03g/L, defoaming agent 0.15g/L, pH=7.10-7.20.
Culture medium D: glucose 4g/L, ammonium sulfate 2.5g/L, dipotassium hydrogen phosphate 0.5g/L, potassium dihydrogen phosphate 0.5g/L, sulphur
Sour magnesium 2g/L, ferrous sulfate 1g/L, calcium carbonate 35g/L, defoaming agent 0.3g/L, Dried Corn Steep Liquor Powder 0.5g/L, sodium glutamate 0.5g/
L, manganese sulfate 0.03g/L, cobalt chloride 0.001g/L, yeast powder 1.0g/L, pH=7.10-7.20.
Culture medium E: glucose 12.6g/L, ammonium sulfate 2.5g/L, dipotassium hydrogen phosphate 0.63g/L, potassium dihydrogen phosphate 0.63g/
L, magnesium sulfate 2.9g/L, ferrous sulfate 0.15g/L, defoaming agent 0.17g/L, Dried Corn Steep Liquor Powder 0.84g/L, sodium glutamate 0.63g/
L, manganese sulfate 0.04g/L, cobalt chloride 0.013g/L, yeast powder 2.1g/L, pH=7.10-7.20.
Culture medium F: ammonium sulfate 3.4g/L, potassium dihydrogen phosphate 0.4g/L, magnesium sulfate 10.4g/L, ferrous sulfate 1.12g/L,
Sodium chloride 2.1g/L, defoaming agent 0.13g/L, calcium chloride dihydrate 0.06g/L, corn pulp 7.5g/L, sodium glutamate 2.4g/L, sulfuric acid
Manganese 0.05g/L, pH=7.10-7.20.
Material 2 in feed supplement 2: ammonium sulfate 5.3g/L, potassium dihydrogen phosphate 2g/L, magnesium sulfate 23g/L, ferrous sulfate 2.1g/L,
Sodium chloride 5.1g/L, defoaming agent 1g/L, calcium chloride dihydrate 0.12g/L, Dried Corn Steep Liquor Powder 1.0g/L, sodium glutamate 6.0g/L, sulphur
Sour manganese 0.12g/L, pH=7.10-7.20.
The sterilising conditions of the above culture medium are 121 DEG C of sterilizing 30min.
Mend sugared sterilising conditions: 60% liquid sugar uses 115.5 ± 0.5 DEG C of sterilizing 25min.
Liquid phosphorus sterilising conditions: 121 ± 1 DEG C of sterilizing 30min of 0.08g/L potassium dihydrogen phosphate.
The ARTP mutagenesis of 1 hydrogenlike silicon ion of embodiment
(1) preparation of bacterial strain seed bacteria suspension:
The hydrogenlike silicon ion for taking -80 DEG C of glycerol tubes to save goes out bacterium germination CF02 (deposit number is CGMCC NO.2458) bacterium solution
100 μ l, are spread evenly across in culture medium A, 32 DEG C of culture 5d.6 single colonies are taken using inoculation shovel after bacterium colony is grown, are inoculated into
It in 50mL/250mL seed bottle containing culture medium B, is sealed using tampon and 2 layers of gauze, 32 DEG C, 220rpm is cultivated for 24 hours, is obtained
Seed bacteria suspension.
(2) preparation of bacteria suspension slide glass:
By metal slide glass in super-clean benchIt is placed in alcolhol burner flame envelope calcination 30s, is put into after cooling primary
Property culture dish or oneself sterilizing glass culture dish in.Above-mentioned cultured seed bacteria suspension 2mL, 4000rpm is taken to be centrifuged 2min, go
Supernatant is added in the physiological saline that mass ratio is 0.8% and is mixed, with diluted to OD600=0.6-0.8 (about 8 times) is dilute
Releasing liquid is that the glycerol that volume ratio is 5% and the physiological saline that mass ratio is 0.8% mix according to the volume ratio of 1:5, is made
Bacteria suspension.It takes 10 μ l bacteria suspensions to be uniformly applied to respectively on 6 slide glasses to prepare sample, pay attention to spreading bacteria suspension uniformly in slide surface
It opens, does 6 every time in parallel.
(3) mutagenesis of bacteria suspension:
ARTP mutagenesis instrument operating room will be moved to equipped with the culture dishes of specimen slides, is taken the photograph son with sterile and be put into 6 slide glasses pair
The hole location answered, shutoff operation room door.Mutagenic condition: mutagenesis power 120W, mutation time 20s, mutagenesis distance 2mm, protection is set
Agent is the glycerol that concentration expressed in percentage by volume is 5%.Click starts to process button, to 6 samples of CF02 seed bacteria suspension successively into
Row ARTP mutagenesis.After sample mutagenesis, slide glass is put into the centrifugation of the 1.5mL equipped with 1mL0.8% physiological saline with aseptic nipper
Guan Zhong, vortex oscillation are mixed 1-3min, the bacterium solution being attached on slide glass are thoroughly eluted in physiological saline, after obtaining mutagenesis
Bacteria suspension.
The screening of the best mutagenic condition of embodiment 2ARTP
To 4 mutagenesis power, mutation time, mutagenesis distance and protective agent concentration parameter designing tests, with bacterial strain lethality
The best mutagenic condition of ARTP mutagenesis hydrogenlike silicon ion is determined for index.When lethality be 85%-95% when, mutagenic condition compared with
It is good, the screening of next step can be carried out, for lethality closer to 90%, mutagenic condition is better.
(1) influence of the different mutagenesis power to lethality: mutation time 20s, mutagenesis distance are 2mm, protective agent is body
The glycerol that product percentage concentration is 5% carries out mutagenesis in the case where mutagenesis power is 100W, 110W, 120W, 130W, 140W respectively, as a result
It is shown in Table 1；
(2) influence of the different mutation times to lethality: mutagenesis power, which is 120W, mutagenesis distance is 2mm, protects protective agent is
The glycerol that concentration expressed in percentage by volume is 5% carries out mutagenesis in the case where mutation time is 10s, 15s, 20s, 25s, 30s respectively, as a result sees
Table 1；
(3) influence of the different mutagenesis distances to lethality: mutagenesis power is 120W, mutation time 20s, protective agent are bodies
The glycerol that product percentage concentration is 5% carries out mutagenesis in the case where mutagenesis distance is 1mm, 2mm, 3mm, 4mm, 5mm respectively, the results are shown in Table
1；
(4) influence of the different protective agent concentration to lethality: mutagenesis power is 120W, mutation time 20s, mutagenesis distance
For 2mm, mutagenesis is carried out in the case where protective agent is the glycerol that concentration expressed in percentage by volume is 3%, 5%, 7%, 10%, 15% respectively, as a result
It is shown in Table 1.
Table 1: influence of the different factors to lethality
As can be seen from Table 1, work as mutagenic condition are as follows: power 100-120W, mutagenesis distance 1-3mm, mutation time 20-
30s, when protective agent is the glycerol that concentration expressed in percentage by volume is 5%-15%, lethality 85%-95%, therefore, under the range
Mutagenic condition is preferable；
When mutagenesis power is 120W, mutation time 20s, mutagenesis distance 2mm, protective agent are that concentration expressed in percentage by volume is 5%
When glycerol, the lethality of hydrogenlike silicon ion is 90.4%, shows that this mutagenic condition is best.
The screening of 3 multiple resistance bacterial strain of embodiment
(1) determination of different resisting substance optimal inhibition concentration
In order to overcome the blindness and non-directiveness of mutagenic and breeding, improves screening efficiency and therefore removed by resistance primary dcreening operation
Major part does not meet the bacterial strain of the production traits.Verifying 4 major class, totally 10 kinds of antibiotic determine different the inhibitory effect of starting strain
The minimum inhibitory concentration of antibiotic.
Antibiotic used is methylnaphthoquinone, kanamycins, roxithromycin, Vitamin K3, adriamycin, Na3N、Na2S, unwrapping wire
Rhzomorph D, benzoic acid, P-hydroxybenzoic acid.
Using starting strain CF02 as subjects, the various concentration gradient of 10 kinds of resisting substances is set, it is cooling to culture medium A
When to 55-60 DEG C, the antibiotic of various concentration is added in solid medium respectively, is mixed, inverted plate.Resistance screening substance is not
2 are shown in Table with concentration gradient setting.It takes 0.1mL to be diluted to 10-6 times of seed liquor, it is flat to be applied to the resisting substance containing various concentration
On plate, each concentration is repeated 3 times, not add the solid medium of resisting substance as control, 32 DEG C of culture 5d, not grow
Minimum inhibitory concentration of the minimum concentration of bacterium colony as the resisting substance.
Table 2: resistance screening substance various concentration gradient
The result shows that methylnaphthoquinone, kanamycins and roxithromycin increase with solvent strength, starting strain growth is not affected by
It influences, therefore later period test is not selected；Finally determine that the minimum inhibitory concentration of other several antibiotic is respectively as follows: Vitamin K3 and is
5mg/L, adriamycin 56.6mg/L, Na3N 1.25mg/L、Na2S 566mg/L, actinomycin D 3.25mg/L, benzoic acid
250mg/L, P-hydroxybenzoic acid 150mg/L.
(2) screening of multiple resistance bacterial strain
Take the bacteria suspension obtained after 1mL mutagenesis in 100mL/500mL seed liquid culture medium, 32 DEG C, 220rpm recovery 2h,
It is diluted to OD600After=0.6-0.8,100 μ l dilutions is taken to be coated on first containing (the 5mg/L dimension life of Co-Q10 analogue
Plain K3 or 55.6mg/L adriamycin) screening flat board on, every kind of resisting substance do 3 it is parallel, 32 DEG C of culture 5d pass through bacterium colony shape
State observation, therefrom picking 1 blackish green, full raised, diameter about 2mm bacterium colony is referred to as R1 as resistant strain.It is with R1
Bacteria suspension after starting strain mutagenesis recovery is coated on added with respiratory chain ubiquinone inhibitor (1.25mg/L Na3N or 566mg/L
Na2S in screening flat board), every kind of resisting substance is repeated 3 times, 32 DEG C of culture 5d, and screening to obtain by same procedure has dual resist
The bacterial strain of property is referred to as R2.It is coated on and is added with as the bacteria suspension after starting strain mutagenesis recovery using R2 bacterial strain by the same way
In the screening flat board of cytotoxic substance (3.25mg/L actinomycin D), repetition does 3 in parallel, and 32 DEG C of culture 5d sieve to obtain 3 weights
Resistant strain is referred to as R3.It is coated on again by the bacteria suspension after starting strain mutagenesis recovery of R3 added with Co-Q10 precursor species
In screening flat board like object (250mg/L benzoic acid, 150mg/L P-hydroxybenzoic acid), every kind of resisting substance does 3 in parallel, and 32
DEG C culture 5d, sieve quadruple resistant strain is referred to as R4.
(3) preservation of resistance primary dcreening operation bacterial strain
Based on 4 weight resistant strains of screening, including other stages, the more excellent bacterial strain single bacterium of screening falls within 50mL/250mL
In seed liquid culture medium, 32 DEG C, 220rpm is cultivated for 24 hours, and by seed liquor: 50% glycerol tube=1:1 volume ratio deposits in -80
DEG C refrigerator carries out cryo-conservation.
Obtained quadruple resistant strain R4 includes the bacterial strain of following name:
CF02-AF3-1, CF02-AF3-19, CF02-AF3-33, CF02-AF3-60, CF02-AF3-66, CF02-AF3-
85, CF02-AF3-103, CF02-AF3-110, CF02-AF3-119, CF02-AF3-126, CF02-AF3-128, CF02-AF3-
154, CF02-AF3-157, CF02-AF3-169, CF02-AF3-171, CF02-AF3-176, CF02-AF3-189, CF02-
AF3-195, CF02-AF3-198, CF02-AF3-199, CF02-AF3-201, CF02-AF3-202, CF02-AF3-230,
CF02-AF3-251, CF02-AF3-253, CF02-AF3-266, CF02-AF3-269, CF02-AF3-272, CF02-AF3-
289, CF02-AF3-292, CF02-AF3-293, CF02-AF3-303, CF02-AF3-312, CF02-AF3-313, CF02-
AF3-315, CF02-AF3-320, CF02-AF3-346, CF02-AF3-389, CF02-AF3-394, CF02-AF3-401,
CF02-AF3-403, CF02-AF3-407, CF02-AF3-429, CF02-AF3-436, CF02-AF3-457, CF02-AF3-
513, CF02-AF3-531, CF02-AF3-537, CF02-AF3-538, CF02-AF3-543, CF02-AF3-544, CF02-
AF3-562, CF02-AF3-587, CF02-AF3-597, CF02-AF3-609, CF02-AF3-610, CF02-AF3-629,
CF02-AF3-635, CF02-AF3-637, CF02-AF3-639, CF02-AF3-645, CF02-AF3-658, CF02-AF3-
661, CF02-AF3-662, CF02-AF3-668, CF02-AF3-674, CF02-AF3-685, CF02-AF3-702, CF02-
AF3-707, CF02-AF3-708, CF02-AF3-718, CF02-AF3-751, CF02-AF3-752, CF02-AF3-770,
CF02-AF3-793, CF02-AF3-801, CF02-AF3-802, CF02-AF3-822, CF02-AF3-840, CF02-AF3-
848, CF02-AF3-853, CF02-AF3-864, CF02-AF3-865, CF02-AF3-877, CF02-AF3-882, CF02-
AF3-894, CF02-AF3-917, CF02-AF3-937
The high flux screening of 4 mutant strain of embodiment
(1) deep-well plates fermented and cultured
The mutant strain R4 for having quadruple resistance that embodiment 3 is obtained is inoculated in the deep-well plates containing culture medium B,
220rmp, 32 DEG C of cultures obtain seed liquor for 24 hours；Take seed liquor into the 24 hole deep-well plates containing 2.7mL culture medium C, 32 DEG C of trainings
Support 72h, each mutant strain do 3 it is parallel, be control with starting strain CF02, inoculum concentration 2%, dress liquid coefficient be 70%,
Shaking speed is 220rpm.
(2) processing of fermentation liquid
By above-mentioned cultured mutant strain fermentation liquid through orifice plate centrifuge, 4500rmp is centrifuged 10min, removes in fermentation liquid
Water phase.300 μ l supernatants are taken, are filtered through vacuum hole panel filter (0.45 μm of filter membrane), each sample filtering three times, finally obtains
1mL filtrate is obtained, the centrifuge tube of 15mL sterilizing-drying is moved it into, 30% hydrogen peroxidase 10 .1mL and 0.1M hydrochloric acid is added into pipe
0.1mL is settled to 10mL with ethyl alcohol.When imbibition, the wall built-up mycelia of flushing hole inner wall is blown and beaten repeatedly, mixes fermentation liquid.Liquid relief
When, it blows and beats repeatedly, wall built-up fermentation liquid in pipette tips is gone in centrifuge tube.Centrifuge tube lid is tightened, after being mixed by inversion, is put into ultrasonic wave
In washer, guarantee that the outer water level of centrifuge tube did not had ethyl alcohol liquid level, ultrasonic 1h in pipe.Centrifuge tube is taken out from ultrasonic cleaner,
It is mixed by inversion, stands about 15min, take upper layer sample liquid 3mL, one-to-one to be added in 96 hole filters, filtrate is through 0.45 μm
Filtrate 1mL is drawn after the filtering of organic phase filter membrane in liquid-phase inlet bottle, with the content of liquid phase detection Co-Q10, i.e. fermentation effect
Valence is control with starting strain CF02, calculates the relative productivity of Co-Q10, screens Co-Q10 relative productivity 6.4% or more
Bacterial strain as high flux screening bacterium.
(3) high performance liquid chromatography measures fermentation titer
Liquid phase testing conditions: mobile phase preparation method is that 350mL dehydrated alcohol and 650mL methanol are mixed loaded on mobile phase
In bottle, ultrasonic 20min exhaust bubble.Operating condition is that HPLC selects UV detector (wavelength=275nm) detection, 10 μ of sample volume
The reversed column of l, 4.6mm*150mm C18,32 DEG C of column temperature, the flow velocity of 1mL/min is detected, runing time about 20min.Detection knot
Fruit sees Fig. 1.
As shown in Figure 1, bacterial strain CF02-AF3-303, CF02-AF3-315, CF02-AF3-394, CF02- have been screened
AF3-407, CF02-AF3-429, CF02-AF3-436, CF02-AF3-531, CF02-AF3-544, CF02-AF3-587 and
CF02-AF3-674 totally 10 plants of mutant strains with quadruple resistance, as high flux screening bacterium, relative productivity is higher,
6.4% or more, wherein the relative productivity of bacterial strain CF02-AF3-674 is maximum, has reached 15.80%.
Screening is verified in the amplification step by step of 5 mutant strain of embodiment
(1) shaking flask is screened
The high flux screening bacterium that Example 4 obtains for 24 hours, is inoculated into containing culture medium B through 32 DEG C of culture medium A plate cultures
In 32 DEG C, 220rpm cultivate for 24 hours, obtain seed liquor, take 5mL seed liquor in be equipped with 60mL fermentation medium C 500mL triangular flask
In, sealed with 8 layers of gauze, the seed liquor of each sample is inoculated with 3 parallel fermentation flasks, 32 DEG C, after 220rpm cultivates 3d, sample into
The detection of row liquid phase, statistical analysis filter out bacterial strain of the fermentation titer more than 528 μ g/mL, are denoted as strains A, are used for subsequent expansion
Fermentation test.As a result see Fig. 2.
(2) 2L tetrad tank ferments
The strains A that shaking flask is screened for 24 hours, is inoculated into containing 32 in culture medium B through 32 DEG C of culture medium A plate cultures
DEG C, 220rpm is cultivated for 24 hours, obtains seed liquor；Fermentation liquid, 121 DEG C of sterilizing 30min are configured according to 60% fermentation charge coefficient；To tank
After temperature drops to 34 DEG C, it is inoculated with 10% seed liquor, 32 DEG C of culture 90h, used medium is culture medium C, and feed supplement 1 is during which added
Feed supplement 3 times.After disappearing and 0h (after kind) sample detection pH, OD, wet thallus content, dissolution phosphorus content, contents of monosaccharides；It is every since 0h
4h sample detection indices and microscopy；Sampling carry out liquid phase detect fermentation titer, filter out fermentation titer 2010 μ g/mL with
On bacterial strain, be denoted as bacterial strain B；As a result see Fig. 3.Fermentation process technique state modulator is shown in Table 2.
Table 2:2L tetrad tank fermentation technology state modulator
(3)20m3Fermentation tank culture
The bacterial strain B that 2L tetrad tank is fermented for 24 hours, is inoculated into containing in culture medium B through 32 DEG C of culture medium A plate cultures
32 DEG C, 220rpm is cultivated for 24 hours, is transferred in the first class seed pot containing culture medium D 32 DEG C, and 220rpm is cultivated for 24 hours, is being transferred to
32 DEG C in secondary seed tank containing culture medium E, 220rpm cultivates 16h, obtains bacterium solution；By bacterium solution culture transferring to 20m3Fermentor,
Used medium is culture medium F, is during which added 2 feed supplement of feed supplement 3 times, and 90h is cultivated.Fermentor major ingredient is dissolved with purified water, stirring
It is uniformly settled to ammonium hydroxide tune pH to 6.6 ± 0.05 after 6600L sterilizes, inoculum concentration 2000L, volume is about 10m after inoculation3。
PH, OD600, sugar, wet thallus, a phosphorus are detected every 4h after fermentation, every 8h detects a potency after 40h, as a result
See Fig. 3 and Fig. 4.Fermentation process technique state modulator is shown in Table 3.
Table 3:20m3Fermentor process parameter control
As seen from Figure 2, it by shaking flask culture primary dcreening operation, is screened from the 10 plant mutant bacterium that high flux screening obtains
Two plants of higher mutant strains of fermentation titer of CF02-AF3-544 and CF02-AF3-674, fermentation titer 528.1 μ g/mL with
On, it is used for subsequent expansion fermentation test.
As can be seen from figs. 3 and 4 by the fermentation of 2L tetrad tank and 20m3Ferment tank culture, CF02-AF3-544 and
The fermentation titer of two plant mutant bacterium of CF02-AF3-674 is apparently higher than former bacterium germination CF02 out, and wherein CF02-AF3-674 is in 20m3Hair
Fermentation titer after fermenting pot culture is maximum, i.e. the content of Co-Q10 is maximum, has reached 2694 μ g/mL, has compared starting strain
The fermentation level of CF02 improves 9.8%.
CF02-AF3-674 hydrogenlike silicon ion mutant strain is named as CPF, has been preserved on October 24th, 2018
Address state General Microbiological Culture preservation administrative center (CGMCC): Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese science
Institute of microbiology, institute；Postcode: 10010, deposit number is CGMCC No.16625.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of high yield Co-Q10 hydrogenlike silicon ion, has been preserved in China General Microbiological culture presevation administrative center, preservation
Number is CGMCC No.16625.
2. the mutagenic breeding method of high yield Co-Q10 hydrogenlike silicon ion according to claim 1, which is characterized in that described
Mutagenic breeding method the following steps are included:
(1) preparation of seed bacteria suspension: activation culture hydrogenlike silicon ion starting strain CF02, picking single colonie, 32 DEG C, 220rpm
Culture for 24 hours, obtains seed bacteria suspension；
The deposit number of the hydrogenlike silicon ion starting strain CF02 is CGMCC NO.2458, is screened by space mutagenesis
It obtains；
(2) preparation of bacteria suspension slide glass: taking seed bacteria suspension, and supernatant is removed in centrifugation, the physiological saline weight for being 0.8% with mass ratio
After outstanding bacterial sediment, with diluted to OD600=0.6-0.8, obtains bacteria suspension, and bacteria suspension is uniformly applied to metal slide glass
Upper obtained ARTP mutagenesis samples；
The physiological saline that the dilution is the glycerol that volume ratio is 5% and mass ratio is 0.8% is mixed according to the volume ratio of 1:5
It closes；
(3) ARTP mutagenesis: mutagenesis is carried out to ARTP mutagenesis samples using ARTP-II type instrument, using 99.99% helium as work
Gas, power 100-120W, mutagenesis distance 1-3mm, mutation time 20-30s, protective agent is that concentration expressed in percentage by volume is 5%-
15% glycerol, after mutagenesis, vortex oscillation 1-3min elutes bacterium solution, the bacteria suspension after obtaining mutagenesis；
(4) multiple resistance screening, high flux screening and amplification verifying step by step the screening of bacterial strain: are carried out to the bacteria suspension after mutagenesis
Screening obtains the high yield Co-Q10 hydrogenlike silicon ion.
3. mutagenic breeding method according to claim 2, which is characterized in that the system of seed bacteria suspension described in step (1)
Standby concrete operations are as follows:
The activation culture hydrogenlike silicon ion starting strain CF02 in culture medium A, 32 DEG C of culture 5d；Picking single colonie, is inoculated in and contains
Have in the seed bottle of culture medium B, 32 DEG C, 220rpm is cultivated for 24 hours, obtains seed bacteria suspension；
The culture medium A includes: yeast powder 1.0-10.0g/L, potassium dihydrogen phosphate 0.1-2.5g/L, magnesium sulfate 1.0-5.0g/
L, ferrous sulfate 0.1-1.0g/L, sodium chloride 0-5.0g/L, ammonium sulfate 1.5-3.5g/L, sodium glutamate 0.5-3.0g/L, corn
Paste dry powder 0.5-3.0g/L, glucose 5-20g/L, agar 15-20g/L, pH=6.5-7.3；
The culture medium B includes: yeast powder 1.0-10.0g/L, potassium dihydrogen phosphate 0.1-2.5g/L, magnesium sulfate 1.0-5.0g/
L, ferrous sulfate 0.1-1.0g/L, sodium chloride 0-5.0g/L, ammonium sulfate 1.5-3.5g/L, sodium glutamate 0.5-3.0g/L, corn
Paste dry powder 0.5-3.0g/L, glucose 5-20g/L, pH=6.5-7.3.
4. mutagenic breeding method according to claim 2, which is characterized in that power described in step (3) is
120W, mutagenesis distance are 2mm, mutation time 20s, protective agent are glycerol that concentration expressed in percentage by volume is 5%.
5. mutagenic breeding method according to claim 3, which is characterized in that the screening of multiple resistance described in step (4)
Include the following steps:
Bacteria suspension rejuvenation after the mutagenesis for taking step (3) to obtain, is inoculated in screening and culturing medium 1 and cultivates, and obtains anti-Co-Q10 knot
The substance resistant strain of structure analog is referred to as R1；
ARTP mutagenesis is carried out by starting strain of R1, rejuvenation is inoculated in screening and culturing medium 2 and cultivates, on R1 underlying resistance
Double resistant strains to anti-respiratory chain ubiquinone inhibitor are named as R2；
ARTP mutagenesis is carried out by starting strain of R2, rejuvenation is inoculated in screening and culturing medium 3 and cultivates, on R2 underlying resistance
Triple resistant strains to anti-cell toxicant are referred to as R3；
ARTP mutagenesis is carried out by starting strain of R3, rejuvenation is inoculated in screening and culturing medium 4 and cultivates, on R3 underlying resistance
Quadruple resistant strain to anti-Co-Q10 precursor analog is referred to as R4；
The screening and culturing medium 1 is that joined Co-Q10 analogue in culture medium A；
The screening and culturing medium 2 is that joined respiratory chain ubiquinone inhibitor in culture medium A；
The screening and culturing medium 3 is that joined cytotoxic substance in culture medium A；
The screening and culturing medium 4 is that joined Co-Q10 precursor analog in culture medium A.
6. mutagenic breeding method according to claim 5, which is characterized in that the Co-Q10 analogue is
5mg/L Vitamin K3 or 55.6mg/L adriamycin；
The respiratory chain ubiquinone inhibitor is 1.25mg/LNa3N or 566mg/L Na2S；
The cytotoxic substance is 3.25mg/L actinomycin D；
The Co-Q10 precursor analog is 250mg/L benzoic acid or 150mg/L P-hydroxybenzoic acid.
7. mutagenic breeding method according to claim 5, which is characterized in that the tool of high flux screening described in step (4)
Steps are as follows for body:
Take the R4 in the deep-well plates containing culture medium B, 220rmp, 32 DEG C of cultures obtain seed liquor for 24 hours；Seed liquor is connect
Kind carry out fermented and cultured in culture medium C, inoculum concentration 2%, dress liquid coefficient be 70%, shaking speed 220rpm, 32 DEG C of trainings
72h is supported, liquid phase detects fermentation liquid potency, screens Co-Q10 relative productivity in 6.4% or more bacterial strain as high flux screening
Bacterium；
The culture medium C includes: Dried Corn Steep Liquor Powder 2.0g/L, sodium glutamate 2.5g/L, ammonium sulfate 3.0g/L, vulcanized sodium
1.5g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 5.0g/L, calcium carbonate 5.0g/L, glucose 20g/L, pH=7.10-7.20；
The liquid phase testing conditions are as follows: wavelength=275nm, the 10 reversed column of μ l, 4.6mm*150mm C18 of sample volume, column temperature 32
DEG C, flow velocity 1mL/min, runing time 20min.
8. mutagenic breeding method according to claim 7, which is characterized in that amplification is verified step by step described in step (4)
Screening step includes: to take to screen by the bacterial strain that high flux screening obtains through shaking flask, the fermentation of 2L tetrad tank, first class seed pot training
It supports, secondary seed tank culture transfers to 20m3Fermentation cylinder for fermentation culture filters out the highest bacterial strain of Co-Q10 yield, i.e.,
For high yield Co-Q10 hydrogenlike silicon ion described in claim 1.
9. mutagenic breeding method according to claim 8, which is characterized in that amplification is verified step by step described in step (4)
Screening specific steps includes:
Shaking flask screening: it takes the bacterial strain obtained by high flux screening through culture medium A plate culture, culture medium B seed culture, then connects
Kind is containing into the shaking flask for having culture medium C, and 32 DEG C, 220rpm is cultivated 3 days, carries out liquid phase detection, filters out fermentation titer in 528 μ
The bacterial strain of g/mL or more, is referred to as strains A；
The fermentation of 2L tetrad tank: the strains A that shaking flask is screened is through culture medium A plate culture, culture medium B seed culture, then connects
Kind is into the 2L tetrad tank containing culture medium C, 32 DEG C of culture 90h, during which supplements feed supplement 1 three times, carries out liquid phase detection, filters out
Bacterial strain of the fermentation titer more than 2010 μ g/mL, is referred to as bacterial strain B；
20m3Fermentor screening: the bacterial strain B that 2L tetrad tank is fermented is through culture medium A plate culture, culture medium B seed culture,
It is cultivated in the first class seed pot of culture medium D for 24 hours, cultivates 16h in the secondary seed tank of culture medium E, transfer to containing culture medium F
20m3During which fermentation cylinder for fermentation culture supplements feed supplement 2 three times, 32 DEG C of culture 90h, carries out liquid phase detection, and screening is weighed
Benefit require 1 described in high yield Co-Q10 hydrogenlike silicon ion；
The culture medium D includes: glucose 4g/L, ammonium sulfate 2.5g/L, dipotassium hydrogen phosphate 0.5g/L, potassium dihydrogen phosphate
0.5g/L, magnesium sulfate 2g/L, ferrous sulfate 1g/L, calcium carbonate 35g/L, defoaming agent 0.3g/L, Dried Corn Steep Liquor Powder 0.5g/L, paddy ammonia
Sour sodium 0.5g/L, manganese sulfate 0.03g/L, cobalt chloride 0.001g/L, yeast powder 1.0g/L, pH=7.10-7.20；
The culture medium E includes: glucose 12.6g/L, ammonium sulfate 2.5g/L, dipotassium hydrogen phosphate 0.63g/L, potassium dihydrogen phosphate
0.63g/L, magnesium sulfate 2.9g/L, ferrous sulfate 0.15g/L, defoaming agent 0.17g/L, Dried Corn Steep Liquor Powder 0.84g/L, sodium glutamate
0.63g/L, manganese sulfate 0.04g/L, cobalt chloride 0.013g/L, yeast powder 2.1g/L, pH=7.10-7.20；
The culture medium F includes: ammonium sulfate 3.4g/L, potassium dihydrogen phosphate 0.4g/L, magnesium sulfate 10.4g/L, ferrous sulfate
1.12g/L, sodium chloride 2.1g/L, defoaming agent 0.13g/L, calcium chloride dihydrate 0.06g/L, corn pulp 7.5g/L, sodium glutamate
2.4g/L, manganese sulfate 0.05g/L, pH=7.10-7.20；
Material 1 in the feed supplement 1 includes: Dried Corn Steep Liquor Powder 6.21g/L, sodium glutamate 1.55g/L, ammonium sulfate 1.55g/L, phosphorus
Acid dihydride potassium 0.62g/L, magnesium sulfate 5.43g/L, ferrous sulfate 0.47g/L, sodium chloride 1.24g/L, manganese sulfate 0.02g/L, chlorine
Change calcium 0.03g/L, defoaming agent 0.15g/L, pH=7.10-7.20；
Material 2 in the feed supplement 2 includes: ammonium sulfate 5.3g/L, potassium dihydrogen phosphate 2g/L, magnesium sulfate 23g/L, ferrous sulfate
2.1g/L, sodium chloride 5.1g/L, defoaming agent 1g/L, calcium chloride dihydrate 0.12g/L, Dried Corn Steep Liquor Powder 1.0g/L, sodium glutamate
6.0g/L, manganese sulfate 0.12g/L, pH=7.10-7.20.
10. application of the hydrogenlike silicon ion described in claim 1 in fermentation preparation of cozymase Q 10.
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