CN109762757B - High-yield coenzyme Q10 rhodobacter sphaeroides and mutation breeding and application thereof - Google Patents
High-yield coenzyme Q10 rhodobacter sphaeroides and mutation breeding and application thereof Download PDFInfo
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Abstract
The invention provides a high-yield coenzyme Q10 rhodobacter sphaeroides and mutation breeding and application thereof, and relates to the field of microorganism mutation breeding. The strain subjected to space mutagenesis screening is taken as a starting strain, the strain is subjected to normal-pressure room-temperature plasma mutagenesis, and multiple resistance screening, high-throughput screening and step-by-step amplification verification screening are carried out to obtain the high-yield coenzyme Q10 rhodobacter sphaeroides which is preserved in the China general microbiological culture collection center with the preservation number of CGMCC No. 16625. The rhodobacter sphaeroides mutant strain obtained by the invention has the length of 20m3Fermentation production verifies that the average titer can reach 2694 mu g/mL, the output of coenzyme Q10 is obviously improved, and in addition, the strain also has quadruple resistance and has wide application prospect.
Description
Technical Field
The invention relates to the field of microorganism mutation breeding, in particular to rhodobacter sphaeroides with high coenzyme Q10 yield and mutation breeding and application thereof.
Background
Coenzyme Q10(CoenzymeQ10, CoQ10), also known as ubiquinone, chemically known as 2, 3-dimethoxy-5-methyl-6-decaisopentenylbenzoquinone, is a retinoid, is present in very low amounts in cells of animals, plants, microorganisms, etc., and can be synthesized in all tissues of the organism. Coenzyme Q10 is an essential coenzyme for generating ATP and a natural antioxidant, participates in cell metabolic activity, can activate and improve human cells, enhances the function of the human immune system, and is widely applied to industries of health care products, foods, cosmetics and the like.
The method for producing coenzyme Q10 raw material mainly comprises biological extraction method, chemical synthesis method and microbial fermentation method, wherein the microbial fermentation method has high biological activity, low raw material cost and capability of improving production capacity by scale-up, and is recognized as a technical process with most advantages and potentials. The strains which produce coenzyme Q10 in nature are mainly Rhodospirillum Molisch (Rhodospirillum Molisch), Agrobacterium (Agrobacterium Conn), Paracoccus (Paracoccus Davis), Pseudomonas (Pseudomonas aeruginosa), Candida (Candida), etc., of which Rhodococcus rhodobacter sphaeroides (Rhodobacter sphaeroides) is one of the major production strains currently used for the large-scale fermentative production of coenzyme Q10. Rhodobacter sphaeroides, while gaining energy for growth and reproduction through the process of cyclic photosynthetic phosphorylation, needs a large amount of accumulated coenzyme Q10 to scavenge free radicals produced by photosynthesis. Usually, the accumulation amount of the wild type microorganism coenzyme Q10 is only (0.8-3.3) mg/g DCW, and the fermentation level is (30-130) mg/L, so that the requirement of industrial large-scale fermentation production cannot be met. At present, the microbial fermentation level of coenzyme Q10 is generally improved through two ways of breeding and modifying high-yield strains and optimizing fermentation conditions and process control, and the main method for obtaining the coenzyme Q10 high-yield strains is to perform mutation breeding by using a traditional physical or chemical method, wherein physical mutation has the advantages of simple method, high safety, low cost, large harvest and the like.
The normal pressure room temperature plasma (ARTP) is called a fourth state of substances except gas, liquid and solid, and plasmas in different thermodynamic states can be generated by changing an excitation mode and a generator structure. The plasma has the characteristics of extremely low ozone concentration and ultraviolet radiation intensity, high safety, environmental friendliness, rapid mutagenesis and the like, and the normal-pressure room-temperature plasma mutagenesis is simple to operate, mild in condition, high in strain mutation rate, and wide in mutation point position and span. The ARTP working gas source type, flow, discharge power, processing time and other conditions are controllable, the intensity of strain mutation and the mutation library capacity can be greatly improved by changing the operating conditions of the instrument, and the ARTP becomes a new method for efficient evolution breeding by combining pressure screening and high-throughput screening technologies. In ARTP mutagenesis, the lethality rate is generally used as an index for screening mutagenesis conditions and the like, and the lethality rate is not too high or too low, and studies show that the closer the lethality rate is to 90%, the better the mutagenesis effect is, and the better the mutagenesis conditions are. At present, Chinese patent publication CN105505915A discloses a method for screening a high-activity formaldehyde-resistant degrading bacteria mutant strain by using ARTP mutagenesis, wherein the mutagenesis conditions are power supply power 115W, working gas flow 10L/min, distance between a plasma emission source and the strain 2mm, operation temperature 23.0-35.0 ℃, and mutagenesis treatment time 0.5-3 min.
Space mutation breeding is widely applied to microorganism breeding in recent years and achieves certain results, party Lei et al (2010) discloses a method for breeding coenzyme Q10 high-yield bacteria by using space mutation, space carrying technology is used for carrying out space mutation breeding on rhodobacter sphaeroides, and finally a mutant strain named Shenzhou6 is obtained, wherein the yield can reach (0.8 +/-0.02) g/L. Although rhodococcus rhodochrous is carried in space for many times and is subjected to space mutagenesis, the strain performance is greatly improved. However, the same mutagenesis mode mainly causes the change of certain gene fixing points of the strains, the strains have certain resistance after being carried for many times, the performance of the bred strains is not obviously improved, the fatigue phenomenon is generated, and the further improvement of the fermentation level of the coenzyme Q10 strains is restricted. Therefore, it is necessary to perform mutagenesis again by using a novel mutagenesis method based on space-mounted mutagenesis breeding, increase mutagenesis sites and screen high-performance mutant strains.
Disclosure of Invention
The invention aims to provide rhodobacter sphaeroides with high coenzyme Q10 yield and mutation breeding and application thereof. ARTP mutagenesis is carried out on the basis of space mutagenesis breeding, so that the problems in the prior art are overcome, and the yield of coenzyme Q10 is improved.
The invention provides a high-yield coenzyme Q10 rhodobacter sphaeroides, the strain number is CPF, the rhodobacter sphaeroides is preserved in China general microbiological culture collection center, and the preservation number is CGMCC No. 16625.
The invention also provides a mutation breeding method of rhodobacter sphaeroides with high coenzyme Q10 yield, which comprises the following steps:
(1) preparation of seed bacteria suspension: activating and culturing rhodobacter sphaeroides starting strain CF02, selecting a single colony, and culturing at 32 ℃ and 220rpm for 24 hours to obtain seed bacteria suspension;
the preservation number of the rhodobacter sphaeroides starting strain CF02 is CGMCC NO.2458, and the rhodobacter sphaeroides starting strain CF02 is obtained by space mutagenesis screening;
(2) preparing a bacterial suspension slide: taking seed bacteria suspension, centrifuging, removing supernatant, resuspending the bacteria precipitate with 0.8% physiological saline by mass ratio, and diluting with diluent to OD600Obtaining bacterial suspension liquid which is 0.6-0.8, and evenly coating the bacterial suspension liquid on a metal slide to prepare an ARTP mutagenesis sample;
the diluent is formed by mixing 5% of glycerol by volume and 0.8% of physiological saline by mass according to the volume ratio of 1: 5;
(3) ARTP mutagenesis: performing mutagenesis on an ARTP mutagenesis sample by using an ARTP-II type instrument, taking 99.99% helium as working gas, taking power supply power of 100-120W, mutagenesis distance of 1-3mm and mutagenesis time of 20-30s, taking glycerol with volume percentage concentration of 5-15% as a protective agent, performing vortex oscillation for 1-3min after mutagenesis, and eluting bacterial liquid to obtain a bacterial suspension after mutagenesis;
preferably, the power of the power supply in the step (3) is 120W, the mutagenesis distance is 2mm, the mutagenesis time is 20s, and the protective agent is glycerol with the volume percentage concentration of 5%.
(4) Screening of strains: and performing multiple resistance screening, high-throughput screening and step-by-step amplification verification screening on the mutagenized bacterial suspension to obtain the high-yield coenzyme Q10 rhodobacter sphaeroides.
Specifically, the seed bacteria suspension of the step (1) is prepared by the following specific operations:
activating and culturing rhodobacter sphaeroides starting strain CF02 on the culture medium A, and culturing for 5d at 32 ℃; selecting a single colony, inoculating the single colony in a seed bottle containing a culture medium B, and culturing at 32 ℃ and 220rpm for 24 hours to obtain seed bacteria suspension;
the culture medium A comprises: 1.0-10.0g/L of yeast powder, 0.1-2.5g/L of monopotassium phosphate, 1.0-5.0g/L of magnesium sulfate, 0.1-1.0g/L of ferrous sulfate, 0-5.0g/L of sodium chloride, 1.5-3.5g/L of ammonium sulfate, 0.5-3.0g/L of sodium glutamate, 0.5-3.0g/L of corn steep liquor dry powder, 5-20g/L of glucose, 15-20g/L of agar and 6.5-7.3 of pH;
the culture medium B comprises: 1.0-10.0g/L of yeast powder, 0.1-2.5g/L of monopotassium phosphate, 1.0-5.0g/L of magnesium sulfate, 0.1-1.0g/L of ferrous sulfate, 0-5.0g/L of sodium chloride, 1.5-3.5g/L of ammonium sulfate, 0.5-3.0g/L of sodium glutamate, 0.5-3.0g/L of corn steep liquor dry powder, 5-20g/L of glucose and 6.5-7.3 of pH.
Preferably, the culture medium A comprises: 5.0g/L of yeast powder, 0.5g/L of monopotassium phosphate, 1.5g/L of magnesium sulfate heptahydrate, 0.1g/L of ferrous sulfate heptahydrate, 2.5g/L of sodium chloride, 2.5g/L of ammonium sulfate, 1.0g/L of sodium glutamate, 2.5g/L of corn steep liquor dry powder, 10g/L of glucose, 15-20g/L of agar and 7.16 of pH;
the culture medium B comprises: 5.0g/L of yeast powder, 0.5g/L of monopotassium phosphate, 1.5g/L of magnesium sulfate heptahydrate, 0.1g/L of ferrous sulfate heptahydrate, 2.5g/L of sodium chloride, 2.5g/L of ammonium sulfate, 1.0g/L of sodium glutamate, 2.5g/L of corn steep liquor dry powder, 10g/L of glucose and 7.16 of pH.
Specifically, the multiple resistance screening in step (4) comprises the following steps:
rejuvenating the bacterial suspension obtained after the mutagenesis in the step (3), inoculating the rejuvenated bacterial suspension into a screening culture medium 1 for culture, and obtaining a single resistant strain resisting the coenzyme Q10 structural analogue which is collectively called R1;
taking R1 as an original strain to carry out ARTP mutagenesis and rejuvenation, inoculating the obtained product in a screening culture medium 2 for culture, and obtaining a double-resistant strain of the anti-respiratory chain ubiquinone inhibitor on the basis of R1 resistance, which is collectively called as R2;
taking R2 as an original strain to carry out ARTP mutagenesis and rejuvenation, inoculating the original strain into a screening culture medium 3 for culture, and obtaining a triple resistant strain of the anti-cytotoxic substance on the basis of R2 resistance, which is collectively called as R3;
taking R3 as an original strain to carry out ARTP mutagenesis, rejuvenation and inoculation in a screening culture medium 4 for culture, and obtaining a quadruple resistance strain resisting coenzyme Q10 precursor analogues on the basis of R3 resistance, which is collectively called as R4;
the screening culture medium 1 is formed by adding coenzyme Q10 structural analogues into a culture medium A;
the screening culture medium 2 is prepared by adding a respiratory chain ubiquinone inhibitor into a culture medium A;
the screening culture medium 3 is prepared by adding a cytotoxic substance into a culture medium A;
the screening culture medium 4 is a culture medium A added with coenzyme Q10 precursor analogues;
the screening culture: repeatedly screening each resistant substance for 3 times, and culturing at 32 deg.C for 5 d;
the method for rejuvenating the mutagenized strain comprises the following steps: 1mL of the bacterial suspension obtained after mutagenesis was taken out of the medium B, recovered at 32 ℃ and 220rpm for 2h, diluted to OD600 of 0.6-0.8, and 100. mu.l of the diluted solution was taken out and coated on a resistant plate for subsequent screening of the resistant plate. And preserving the screened better strains to construct a strain mutation library.
The strain preservation method comprises the following steps: the selected single colony is cultured in a seed liquid culture medium of 50mL/250mL at 32 ℃ and 220rpm for 24h to obtain a seed liquid, and the seed liquid is stored in a refrigerator at the temperature of-80 ℃ for low-temperature storage according to the volume ratio of the seed liquid to 50% glycerol of 1: 1.
Preferably, the structural analog of the coenzyme Q10 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 coenzyme Q10 precursor analogue is 250mg/L benzoic acid or 150mg/L p-hydroxybenzoic acid.
Specifically, the high-throughput screening described in step (4) comprises the following steps:
culturing the R4 in a deep-well plate containing a culture medium B at 220rmp at 32 ℃ for 24h to obtain a seed solution; inoculating the seed solution into a culture medium C for fermentation culture, wherein the inoculation amount is 2%, the liquid filling coefficient is 70%, the rotating speed of a shaking table is 220rpm, the culture is carried out for 72 hours at 32 ℃, the titer of fermentation liquor is detected by a liquid phase, and strains with the relative yield of coenzyme Q10 of more than 6.4% are screened as high-throughput screening bacteria;
the culture medium C comprises: 2.0g/L of corn steep liquor dry powder, 2.5g/L of sodium glutamate, 3.0g/L of ammonium sulfate, 1.5g/L of sodium sulfide, 0.5g/L of monopotassium phosphate, 5.0g/L of magnesium sulfate, 5.0g/L of calcium carbonate, 20g/L of glucose and 7.10-7.20 of pH;
the liquid phase detection conditions are as follows: wavelength 275nm, sample size 10. mu.l, 4.6mm 150mm C18 reverse column, column temperature 32 ℃, flow rate 1mL/min, run time 20 min.
Specifically, the method for detecting the titer of the fermentation liquor of the deep-hole plate comprises the following steps: centrifuging the cultured mutant strain fermentation liquid by a pore plate centrifuge at 4500rmp for 10min, removing water phase in the fermentation liquid, taking 300 mu l of supernatant, filtering the supernatant by a vacuum pore plate filter (0.45 mu M filter membrane) for three times to finally obtain 1mL of filtrate, transferring the filtrate into a sterilized and dried centrifuge tube, adding 0.1mL of 30% hydrogen peroxide and 0.1mL of 0.1M hydrochloric acid into the tube, diluting to 10mL with ethanol, washing wall-hung mycelia on the inner wall of the pore during imbibition, repeatedly blowing and beating, uniformly mixing the fermentation liquid, repeatedly blowing and beating during pipetting, transferring the wall-hung fermentation liquid in the gun head into the centrifuge tube, reversely and uniformly mixing, putting the centrifuge tube into an ultrasonic cleaner, performing ultrasonic treatment for 1h, taking out the centrifuge tube from the ultrasonic cleaner, reversely and uniformly mixing, standing for 15min, taking 300 mu l of upper layer sample liquid, adding into a 96 pore filter, filtering the filtrate by a 0.45 mu M organic phase filter membrane, sucking 1mL of the filtrate into a liquid phase sample bottle, detection was performed with the liquid phase.
Specifically, the step of step (4) of stepwise amplification verification screening includes: screening the high-throughput screening strain obtained in claim 5 by shaking a flask, fermenting in a 2L tetrad tank, culturing in a first seeding tank, culturing in a second seeding tank, and transferring to 20m3Fermenting and culturing in a fermentation tank, and screening to obtain high-yield coenzyme Q10 rhodobacter sphaeroides.
More specifically, the step of step (4) of progressive amplification verification screening includes:
and (3) screening in a shaking flask: culturing high-throughput screening bacteria by a culture medium A plate, culturing a culture medium B seed, inoculating into a shake flask containing a culture medium C, culturing at 32 ℃ and 220rpm for 3 days, performing liquid phase detection, and screening out strains with fermentation titer of more than 528 mug/mL, which are collectively called as a strain A;
2L fermentation in a tetrad tank: carrying out plate culture on the strain A obtained by screening in a shake flask through a culture medium A, carrying out seed culture on a culture medium B, inoculating the strain A into a 2L four-way tank containing a culture medium C, culturing for 90h at 32 ℃, supplementing and supplementing materials for 1 time during the culture for three times, carrying out liquid phase detection, and screening out the strains with the fermentation titer of more than 2010 mu g/mL, wherein the strains are collectively called as a strain B;
20m3screening a fermentation tank: culturing strain B obtained by 2L fermentation in tetrad tank with culture medium A plate, culturing with culture medium B seed, culturing in first-stage seed tank of culture medium D for 24 hr, culturing in second-stage seed tank of culture medium E for 16 hr, transferring to 20m containing culture medium F3Fermenting and culturing in a fermentation tank, supplementing materials for 2 times during the fermentation, culturing for 90h, performing liquid phase detection, and screening to obtain high-yield coenzyme Q10 rhodobacter sphaeroides.
Specifically, the culture medium D comprises: 4g/L of glucose, 2.5g/L of ammonium sulfate, 0.5g/L of dipotassium phosphate, 0.5g/L of potassium dihydrogen phosphate, 2g/L of magnesium sulfate, 1g/L of ferrous sulfate, 35g/L of calcium carbonate, 0.3g/L of defoaming agent, 0.5g/L of corn steep liquor dry powder, 0.5g/L of sodium glutamate, 0.03g/L of manganese sulfate, 0.001g/L of cobalt chloride, 1.0g/L of yeast powder and 7.10-7.20 of pH;
the culture medium E comprises: 12.6g/L of glucose, 2.5g/L of ammonium sulfate, 0.63g/L of dipotassium phosphate, 0.63g/L of monopotassium phosphate, 2.9g/L of magnesium sulfate, 0.15g/L of ferrous sulfate, 0.17g/L of defoaming agent, 0.84g/L of corn steep liquor dry powder, 0.63g/L of sodium glutamate, 0.04g/L of manganese sulfate, 0.013g/L of cobalt chloride, 2.1g/L of yeast powder and 7.10-7.20 of pH;
the culture medium F comprises: 3.4g/L of ammonium sulfate, 0.4g/L of monopotassium phosphate, 10.4g/L of magnesium sulfate, 1.12g/L of ferrous sulfate, 2.1g/L of sodium chloride, 0.13g/L of defoaming agent, 0.06g/L of calcium chloride dihydrate, 7.5g/L of corn steep liquor, 2.4g/L of sodium glutamate, 0.05g/L of manganese sulfate and pH value of 7.10-7.20;
the material 1 in the feed supplement 1 comprises: 6.21g/L of corn steep liquor dry powder, 1.55g/L of sodium glutamate, 1.55g/L of ammonium sulfate, 0.62g/L of monopotassium phosphate, 5.43g/L of magnesium sulfate, 0.47g/L of ferrous sulfate, 1.24g/L of sodium chloride, 0.02g/L of manganese sulfate, 0.03g/L of calcium chloride, 0.15g/L of defoaming agent and 7.10-7.20 of pH value;
the feed 2 in the feed supplement 2 comprises: 5.3g/L of ammonium sulfate, 2g/L of monopotassium phosphate, 23g/L of magnesium sulfate, 2.1g/L of ferrous sulfate, 5.1g/L of sodium chloride, 1g/L of defoaming agent, 0.12g/L of calcium chloride dihydrate, 1.0g/L of corn steep liquor dry powder, 6.0g/L of sodium glutamate, 0.12g/L of manganese sulfate and 7.10-7.20 of pH value.
The sterilization conditions of the culture medium are 121 ℃ for 30 min; sugar supplementing and sterilizing conditions: sterilizing 60% liquid sugar at 115.5 + -0.5 deg.C for 25 min; liquid phosphorus sterilization conditions: 0.08g/L potassium dihydrogen phosphate is sterilized at 121 +/-1 ℃ for 30 min.
The invention also provides application of the rhodobacter sphaeroides with high coenzyme Q10 yield in fermentation production of coenzyme Q10;
compared with the prior art, the invention has the advantages that:
1. the invention takes the strains subjected to space mutagenesis screening as the basis, carries out normal pressure room temperature plasma (ARTP) mutagenesis on the strains, and obtains a high-yield coenzyme Q10 rhodobacter sphaeroides which is preserved in the China general microbiological culture collection center with the preservation number of CGMCC No.16625 through multiple resistance screening, high-throughput screening and stage-by-stage amplification verification screening. The strain is 20m3The fermentation production verifies that the average titer can reach 2694 mu g/mL, the fermentation level of the starting strain CF02 is improved by 9.8 percent, the yield of coenzyme Q10 is obviously improved, and in addition, the strain also has quadruple resistance and has wide application prospect.
2. The mutation screening method provided by the invention optimizes the ARTP mutation conditions, is more suitable for the mutation of rhodobacter sphaeroides, and expands the ARTP mutation breeding range; the screening of multiple resistant strains is increased, so that the strains obtain multiple resistance; the device adopts 24-hole deep hole plates, hole plate centrifugation, vacuum plate filtration and the like, so that the cost of screening materials is obviously reduced, and the working efficiency and the screening flux are improved; shaking the flask, 2L tetrad tank, 20m3Progressive amplification of fermentation tankThe strain is verified to be re-screened, the screening of the strain is completed comprehensively and systematically, and the obtained strain has good genetic stability and important economic value and social significance.
Drawings
FIG. 1 is a graph of the results of high throughput screening in example 4;
FIG. 2 is a histogram of fermentation titers from shake flask fermentations in example 5;
FIG. 3 shows a 2L four-tank system and 20m in example 53A histogram of fermentation titers from the fermentor;
FIG. 4 shows 20m of the mutant strain CPF and the starting strain CF02 in example 53Titer of fermentation process in fermenter is shown.
Detailed Description
The following description of the embodiments is only intended to aid in the understanding of the method of the invention and its core ideas. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention. The following description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The examples do not show the specific techniques or conditions, and the techniques or conditions are described in the literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruker et al, Huang Petang et al) or according to the product instructions.
The media formulations used in the specific examples are as follows:
culture medium A: 5.0g/L of yeast powder, 0.5g/L of monopotassium phosphate, 1.5g/L of magnesium sulfate heptahydrate, 0.1g/L of ferrous sulfate heptahydrate, 2.5g/L of sodium chloride, 2.5g/L of ammonium sulfate, 1.0g/L of sodium glutamate, 2.5g/L of corn steep liquor dry powder, 10g/L of glucose, 15-20g/L of agar and 7.16 of pH.
And (3) a culture medium B: agar was not added, and the other was in accordance with the plate medium formulation.
And (3) a culture medium C: 2.0g/L of corn steep liquor dry powder, 2.5g/L of sodium glutamate, 3.0g/L of ammonium sulfate, 1.5g/L of sodium sulfide, 0.5g/L of monopotassium phosphate, 5.0g/L of magnesium sulfate, 5.0g/L of calcium carbonate, 20g/L of glucose and 7.10-7.20 of pH value.
Feed 1 in feed 1: 6.21g/L of corn steep liquor dry powder, 1.55g/L of sodium glutamate, 1.55g/L of ammonium sulfate, 0.62g/L of monopotassium phosphate, 5.43g/L of magnesium sulfate, 0.47g/L of ferrous sulfate, 1.24g/L of sodium chloride, 0.02g/L of manganese sulfate, 0.03g/L of calcium chloride, 0.15g/L of defoaming agent and 7.10-7.20 of pH value.
And (3) a culture medium D: 4g/L of glucose, 2.5g/L of ammonium sulfate, 0.5g/L of dipotassium hydrogen phosphate, 0.5g/L of potassium dihydrogen phosphate, 2g/L of magnesium sulfate, 1g/L of ferrous sulfate, 35g/L of calcium carbonate, 0.3g/L of defoaming agent, 0.5g/L of corn steep liquor dry powder, 0.5g/L of sodium glutamate, 0.03g/L of manganese sulfate, 0.001g/L of cobalt chloride, 1.0g/L of yeast powder and 7.10-7.20 of pH.
And (3) a culture medium E: 12.6g/L of glucose, 2.5g/L of ammonium sulfate, 0.63g/L of dipotassium phosphate, 0.63g/L of monopotassium phosphate, 2.9g/L of magnesium sulfate, 0.15g/L of ferrous sulfate, 0.17g/L of defoaming agent, 0.84g/L of corn steep liquor dry powder, 0.63g/L of sodium glutamate, 0.04g/L of manganese sulfate, 0.013g/L of cobalt chloride, 2.1g/L of yeast powder and 7.10-7.20 of pH.
And (3) a culture medium F: 3.4g/L of ammonium sulfate, 0.4g/L of monopotassium phosphate, 10.4g/L of magnesium sulfate, 1.12g/L of ferrous sulfate, 2.1g/L of sodium chloride, 0.13g/L of defoaming agent, 0.06g/L of calcium chloride dihydrate, 7.5g/L of corn steep liquor, 2.4g/L of sodium glutamate, 0.05g/L of manganese sulfate and pH value of 7.10-7.20.
Feed 2 in feed 2: 5.3g/L of ammonium sulfate, 2g/L of monopotassium phosphate, 23g/L of magnesium sulfate, 2.1g/L of ferrous sulfate, 5.1g/L of sodium chloride, 1g/L of defoaming agent, 0.12g/L of calcium chloride dihydrate, 1.0g/L of corn steep liquor dry powder, 6.0g/L of sodium glutamate, 0.12g/L of manganese sulfate and 7.10-7.20 of pH value.
The above culture medium was sterilized at 121 deg.C for 30 min.
Sugar supplementing and sterilizing conditions: 60% liquid sugar is sterilized at 115.5 + -0.5 deg.C for 25 min.
Liquid phosphorus sterilization conditions: 0.08g/L potassium dihydrogen phosphate is sterilized at 121 +/-1 ℃ for 30 min.
EXAMPLE 1 ARTP mutagenesis of rhodobacter sphaeroides
(1) Preparing a strain seed bacteria suspension:
taking 100 μ l of rhodobacter sphaeroides strain CF02 (with preservation number of CGMCC NO.2458) stored in glycerol tube at-80 deg.C, uniformly coating on the culture medium A, and culturing at 32 deg.C for 5 d. And after the colonies grow out, using an inoculating shovel to take 6 single colonies, inoculating the single colonies into a 50mL/250mL seed bottle containing a culture medium B, sealing the seed bottle by using a cotton plug and 2 layers of gauze, and culturing the seed bottle at 32 ℃ and 220rpm for 24 hours to obtain a seed bacteria suspension.
(2) Preparing a bacterial suspension slide:
carrying metal slide in super clean bench
Burning with outer flame of alcohol lamp for 30s, cooling, and placing into disposable culture dish or sterilized glass culture dish. Centrifuging 2mL of the cultured seed bacteria suspension at 4000rpm for 2min, removing supernatant, adding 0.8% physiological saline water, mixing, and diluting with diluent to OD600The dilution is prepared by mixing 5% glycerol by volume and 0.8% physiological saline by mass according to the volume ratio of 1:5 to prepare bacterial suspension. Samples were prepared by spreading 10 μ l of the bacterial suspension evenly onto 6 slides, each time in 6 replicates, with care to spread the bacterial suspension evenly across the slide surface.
(3) Mutagenesis of the bacterial suspension:
the petri dish with the sample slide was moved to the ARTP mutagen operating room, 6 slides were placed in the corresponding wells with sterile forceps, and the door of the operating room was closed. Setting mutagenesis conditions: the mutagenesis power is 120W, the mutagenesis time is 20s, the mutagenesis distance is 2mm, and the protective agent is glycerol with the volume percentage concentration of 5 percent. The start treatment button was clicked and 6 samples of the CF02 seed suspension were subjected to ARTP mutagenesis in sequence. After the mutagenesis of the sample is finished, placing the slide glass into a 1.5mL centrifuge tube filled with 1mL of 0.8% physiological saline by using sterile forceps, carrying out vortex oscillation and uniform mixing for 1-3min, and completely eluting bacterial liquid attached to the slide glass into the physiological saline to obtain a mutagenized bacterial suspension.
Example 2 screening of optimal mutagenesis conditions for ARTP
4 parameters of mutagenesis power, mutagenesis time, mutagenesis distance and protective agent concentration are designed and tested, and the optimal mutagenesis condition of the ARTP mutagenesis rhodobacter sphaeroides is determined by taking the strain lethality as an index. When the lethality is 85% -95%, the mutagenesis condition is better, the next screening can be carried out, and the closer the lethality is to 90%, the better the mutagenesis condition is.
(1) Effect of different mutagenesis powers on lethality: the mutagenesis time is 20s, the mutagenesis distance is 2mm, the protective agent is glycerol with the volume percentage concentration of 5 percent, and the mutagenesis is respectively carried out under the mutagenesis power of 100W, 110W, 120W, 130W and 140W, and the result is shown in the table 1;
(2) effect of different mutagenesis times on lethality: the mutagenesis power is 120W, the mutagenesis distance is 2mm, the protective agent is glycerol with the volume percentage concentration of 5 percent, and the mutagenesis is respectively carried out under the mutagenesis time of 10s, 15s, 20s, 25s and 30s, and the result is shown in the table 1;
(3) effect of different mutagenesis distances on lethality: the mutagenesis power is 120W, the mutagenesis time is 20s, the protective agent is glycerol with the volume percentage concentration of 5 percent, and the mutagenesis is respectively carried out under the mutagenesis distances of 1mm, 2mm, 3mm, 4mm and 5mm, and the result is shown in the table 1;
(4) effect of different protectant concentrations on lethality: the mutagenesis power was 120W, the mutagenesis time was 20s, the mutagenesis distance was 2mm, and the mutagenesis was performed with 3%, 5%, 7%, 10%, 15% glycerol as the protective agent by volume, respectively, and the results are shown in Table 1.
Table 1: effect of different factors on lethality
As can be seen from table 1, when the mutagenesis conditions were: the power of the power source is 100-;
when the mutagenesis power is 120W, the mutagenesis time is 20s, the mutagenesis distance is 2mm, and the protective agent is glycerol with the volume percentage concentration of 5 percent, the lethality of rhodobacter sphaeroides is 90.4 percent, which indicates that the mutagenesis condition is optimal.
Example 3 selection of multiple resistant strains
(1) Determination of optimal inhibitory concentrations of different resistant substances
In order to overcome the blindness and the non-tropism of mutagenesis breeding and improve the screening efficiency, most strains which do not accord with the production traits are removed through resistance primary screening. And (3) verifying the inhibition effect of 10 antibiotics in 4 classes on the original strain, and determining the minimum inhibition concentration of different antibiotics.
The antibiotic is menadione, kanamycin, roxithromycin, vitamin K3, adriamycin, and Na3N、Na2S, actinomycin D, benzoic acid and p-hydroxybenzoic acid.
Setting different concentration gradients of 10 resistant substances by taking a starting strain CF02 as a test object, respectively adding antibiotics with different concentrations into a solid culture medium when a culture medium A is cooled to 55-60 ℃, uniformly mixing, and pouring a flat plate. The different concentration gradient settings of the resistance selection substances are shown in Table 2. 0.1mL of the seed solution diluted 10-6 times was applied to plates containing different concentrations of the resistant substance, each concentration was repeated 3 times, the solid medium without the resistant substance was used as a control, and the medium was incubated at 32 ℃ for 5 days, and the lowest concentration at which colonies did not grow was used as the minimum inhibitory concentration of the resistant substance.
Table 2: gradient of different concentrations of resistance screening substance
The results show that the growth of the original strain is not influenced as the concentration of the solvent is increased by the menaquinone, the kanamycin and the roxithromycin, so that later-period tests are not selected; finally, the minimum inhibitory concentrations of the other antibiotics were determined as follows: vitamin K3 is 5mg/L, adriamycin 56.6mg/L, Na3N 1.25mg/L、Na2S566 mg/L, actinomycin D3.25 mg/L, benzoic acid 250mg/L and p-hydroxybenzoic acid 150 mg/L.
(2) Screening for multiple resistant strains
Taking 1mL of bacterial suspension obtained after mutagenesis, putting the bacterial suspension in a seed liquid culture medium of 100mL/500mL, recovering for 2h at 32 ℃, 220rpm, and diluting to OD600After 0.6-0.8, 100. mu.l of the dilution was first spread on a selection plate containing a coenzyme Q10 structural analogue (5mg/L vitamin K3 or 55.6mg/L doxorubicin), 3 replicates of each resistant substance were cultured at 32 ℃ for 5 days, and 1 dark green colony with a full bulge and a diameter of about 2mm was selected from the colonies as a resistant strain, which was collectively referred to as R1 by colony morphology observation. The bacterial suspension obtained after the mutagenesis recovery by using R1 as the starting strain is coated with respiratory chain ubiquinone inhibitor (1.25mg/L Na)3N or 566mg/L Na2S), repeating the culture for 5 days at 32 ℃ for 3 times for each resistant substance on the screening plate, and screening the strains with dual resistance according to the same method to obtain a strain which is collectively called R2. According to the same method, bacterial suspension obtained after the R2 strain is used as an original strain and subjected to mutagenesis recovery is coated on a screening plate added with a cytotoxic substance (3.25mg/L actinomycin D), 3 parallel operations are repeatedly carried out, the culture is carried out for 5 days at 32 ℃, and 3 resistant strains are screened and are collectively called R3. And then, taking R3 as an original strain, performing mutagenesis recovery, coating the bacterial suspension on a screening plate added with coenzyme Q10 precursor analogues (250mg/L benzoic acid and 150mg/L p-hydroxybenzoic acid), performing parallel culture on 3 resistant substances at 32 ℃ for 5 days, and screening to obtain quadruple resistant strains which are collectively called R4.
(3) Preservation of resistant Primary Sieve Strain
Selecting 4 resistant strains as main strains, and culturing single colonies of better strains selected in other stages in a seed liquid culture medium of 50mL/250mL at 32 ℃ and 220rpm for 24h, and storing the strains in a refrigerator of-80 ℃ for low-temperature storage according to the volume ratio of the seed liquid to 50% glycerol tube of 1: 1.
The resulting quadruple resistant strain R4 included the following named strains:
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
example 4 high throughput screening of mutant strains
(1) Deep-well plate fermentation culture
The mutant strain R4 with quadruple resistance obtained in example 3 is inoculated into a deep-well plate containing a culture medium B, and is cultured for 24 hours at the temperature of 32 ℃ at 220rmp to obtain a seed solution; taking the seed solution to a 24-hole deep-well plate containing 2.7mL of culture medium C, culturing for 72h at 32 ℃, carrying out 3 parallels on each mutant strain, taking the original strain CF02 as a control, wherein the inoculation amount is 2%, the liquid loading coefficient is 70%, and the rotating speed of a shaker is 220 rpm.
(2) Treatment of fermentation broths
And (3) centrifuging the cultured mutant strain fermentation liquor for 10min at 4500rmp by using a pore plate centrifuge, and removing a water phase in the fermentation liquor. Mu.l of the supernatant was filtered through a vacuum well filter (0.45 μ M filter) and each sample was filtered three times to obtain 1mL of filtrate, which was transferred to a 15mL sterile dry centrifuge tube, and 0.1mL of 30% hydrogen peroxide and 0.1mL of 0.1M hydrochloric acid were added to the tube and the volume was adjusted to 10mL with ethanol. And (3) flushing the wall-hung mycelia on the inner wall of the hole during liquid suction, repeatedly blowing and beating, and uniformly mixing the fermentation liquid. And repeatedly blowing and beating during liquid transferring, and transferring the wall-hung fermentation liquid in the gun head into a centrifuge tube. Screwing down the centrifugal tube cover, reversing and uniformly mixing, putting into an ultrasonic cleaner, and performing ultrasonic treatment for 1h to ensure that the water level outside the centrifugal tube is not higher than the ethanol liquid level inside the tube. Taking out the centrifuge tube from the ultrasonic cleaner, reversing and uniformly mixing, standing for about 15min, taking 3mL of upper-layer sample liquid, adding the upper-layer sample liquid into a 96-hole filter in a one-to-one correspondence manner, filtering the filtrate by using a 0.45-micrometer organic phase filter membrane, absorbing 1mL of the filtrate into a liquid-phase sample injection bottle, detecting the content of coenzyme Q10 by using a liquid phase, namely fermentation titer, calculating the relative yield of coenzyme Q10 by using a starting strain CF02 as a control, and screening a strain with the relative yield of coenzyme Q10 of more than 6.4% as a high-throughput screening strain.
(3) High performance liquid chromatography for measuring fermentation titer
Liquid phase detection conditions: the preparation method of the mobile phase comprises the steps of mixing 350mL of absolute ethyl alcohol and 650mL of methanol, uniformly filling the mixture into a mobile phase bottle, and performing ultrasonic treatment for 20min to remove bubbles. The working conditions were HPLC detection with a selective UV detector (wavelength 275nm), a sample volume of 10. mu.l, a 4.6mm 150mm C18 reverse column, a column temperature of 32 ℃ and a flow rate of 1mL/min, and a running time of about 20 min. The results are shown in FIG. 1.
As can be seen from FIG. 1, the screened strains CF02-AF3-303, CF02-AF3-315, CF02-AF3-394, CF02-AF3-407, CF02-AF3-429, CF02-AF3-436, CF02-AF3-531, CF02-AF3-544, CF02-AF3-587 and CF02-AF3-674 share 10 strains with quadruple resistance, and as high-throughput screening strains, the relative yield is high and is more than 6.4%, wherein the relative yield of the strains CF02-AF3-674 is the maximum and reaches 15.80%.
Example 5 stepwise amplification of mutant strains for validation screening
(1) Shake flask screening
Culturing the high-throughput screening bacteria obtained in the example 4 by a plate of a culture medium A at 32 ℃ for 24h, inoculating the high-throughput screening bacteria into a culture medium B at 32 ℃, culturing at 220rpm for 24h to obtain a seed solution, placing 5mL of the seed solution into a 500mL triangular flask containing 60mL of a fermentation culture medium C, sealing the flask by 8 layers of gauze, inoculating 3 parallel fermentation flasks into the seed solution of each sample, culturing at 32 ℃, at 220rpm for 3d, sampling, performing liquid phase detection, performing statistical analysis, screening out a strain with the fermentation titer of more than 528 mu g/mL, and marking as a strain A for a subsequent enlarged fermentation test. The results are shown in FIG. 2.
(2)2L four-tank fermentation
Culturing the strain A obtained by screening in a shake flask through a culture medium A plate at 32 ℃ for 24h, inoculating the strain A into a culture medium B at 32 ℃, and culturing at 220rpm for 24h to obtain a seed solution; preparing fermentation liquor according to 60% fermentation charging coefficient, and sterilizing at 121 deg.C for 30 min; after the temperature of the tank is reduced to 34 ℃, inoculating 10 percent of seed liquid, culturing for 90 hours at 32 ℃, wherein the used culture medium is a culture medium C, and 1 supplementary material is added during the culture period for 3 times. Sampling after digestion and 0h (after seeding) to detect pH, OD, wet thallus content, dissolved phosphorus content and monosaccharide content; sampling every 4h from 0h to detect each index and performing microscopic examination; sampling, carrying out liquid phase detection on fermentation titer, screening out a strain with the fermentation titer being more than 2010 mu g/mL, and marking as a strain B; the results are shown in FIG. 3. The control of the fermentation process parameters is shown in Table 2.
Table 2: 2L tetrad tank fermentation process parameter control
(3)20m3Cultivation in fermenter
Culturing a strain B obtained by fermenting a 2L tetrad tank for 24h at 32 ℃ through a plate of a culture medium A, inoculating the strain B into the culture medium B for 24h at 32 ℃ and 220rpm, transferring the strain B into a primary seed tank containing a culture medium D for 24h at 32 ℃ and 220rpm, and culturing the strain B for 16h at 32 ℃ and 220rpm in a secondary seed tank containing a culture medium E to obtain a bacterial liquid; transferring the bacterial liquid to 20m3The fermenter is fed with medium F, during which period the feed 2 is addedThe feed was added 3 times and cultured for 90 h. Dissolving the main material in purified water, stirring, diluting to 6600L, sterilizing, adjusting pH to 6.6 + -0.05 with ammonia water, inoculating to 2000L, and inoculating to volume of 10m3。
After fermentation, pH, OD600, sugar, wet bacteria and phosphorus were measured every 4h, and after 40h, titer was measured every 8h, and the results are shown in FIGS. 3 and 4. The control of the fermentation process parameters is shown in Table 3.
Table 3: 20m3Fermentation tank process parameter control
As can be seen from FIG. 2, after primary screening by shake flask culture, two mutant strains with higher fermentation titer, namely CF02-AF3-544 and CF02-AF3-674, were screened from 10 mutant strains obtained by high-throughput screening, and the fermentation titer was 528.1 μ g/mL or more, and the strains were used in the subsequent extended fermentation test.
As can be seen from FIGS. 3 and 4, 2L of the fermentation broth was fermented in a tetrad tank at 20m3The fermentation titer of the two mutant bacteria of CF02-AF3-544 and CF02-AF3-674 is obviously higher than that of the original mutant bacteria CF02, wherein CF02-AF3-674 is at 20m3The fermentation titer after fermentation culture in a fermentation tank is the maximum, namely the content of coenzyme Q10 is the maximum, which reaches 2694 mu g/mL and is improved by 9.8 percent compared with the fermentation level of the starting strain CF 02.
The mutant strain of the rhodobacter sphaeroides CF02-AF3-674 is named as CPF, and is preserved in the China general microbiological culture Collection center (CGMCC) at 10-24 months in 2018: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; and E, postcode: 10010, preservation number is CGMCC No. 16625.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (2)
1. A rhodobacter sphaeroides with high-yield coenzyme Q10 is preserved in China general microbiological culture collection center with the preservation number of CGMCC No. 16625.
2. Use of rhodobacter sphaeroides according to claim 1 for the fermentative production of coenzyme Q10.
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