Rhodobacter sphaeroides and application thereof in de novo synthesis of oxidized coenzyme Q10

Abstract

The invention relates to the technical field of biology, in particular to rhodobacter sphaeroides and application of the rhodobacter sphaeroides in de novo synthesis of oxidized coenzyme Q10. The invention provides Rhodobacter sphaeroides INGIA-NM002 with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.32823. The oxidized coenzyme Q10 is produced by fermenting a substrate (glucose or glycerol) by adopting the strain, so that the yield and the efficiency of industrial production are improved, and the market demand is met.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a spherical red bacterium and an application thereof in de novo synthesis of oxidized coenzyme Q10. Background Art

[0002] Coenzyme Q10 (CoQ10), chemically named 2,3-dimethoxy-5-methyl-6-decyl isopentenyl benzoquinone, is widely distributed in the biomembranes of various organisms and is an important electron acceptor in the cell respiratory chain. Coenzyme Q10 is a natural antioxidant and cell metabolism activator that can prevent and treat a variety of diseases. It is also widely used in fighting fatigue, treating heart failure, and delaying skin aging, which has further expanded the demand at home and abroad.

[0003] At present, coenzyme Q10 can be prepared by three methods: natural extraction, chemical synthesis and biosynthesis. Among them, the natural extraction method is limited by raw materials and sources. The content of coenzyme Q10 in animals and plants is low, and large-scale production is subject to certain restrictions. The products produced are high in cost and expensive. The product of the chemical synthesis method is a mixture of cis and trans isomers with low biological activity. Coenzyme Q10 produced by biosynthesis, i.e. fermentation, has low cost, no optical isomers, high biological activity, and good large-scale production application effect. Therefore, biosynthesis is the mainstream production process in industrial production.

[0004] There have been many research reports on the fermentation process of coenzyme Q10 produced by Aspergillus fumigatus, Rhodobacter penetrating membrane, Agrobacterium tumefaciens, Rhodobacter sphaeroides, etc. At present, the strains used for coenzyme Q10 fermentation production still need to be further improved to improve the efficiency of industrial production and meet market demand. Summary of the invention

[0005] In view of this, the present invention provides a sphaeroides Rhodobacter and its use in the de novo synthesis of oxidized coenzyme Q10, which can be used to de novo synthesize oxidized coenzyme Q10 based on glucose or glycerol to improve the output and efficiency of industrial production to meet market demand.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides Rhodobacter sphaeroides, whose deposit number is CGMCC No.32823.

[0008] The present invention also provides the use of the Rhodobacter sphaeroides in synthesizing oxidized coenzyme Q10.

[0009] In some specific embodiments of the present invention, the synthesis of the above application includes de novo synthesis;

[0010] The de novo synthesis includes synthesis using glucose or glycerol as a substrate.

[0011] The present invention also provides a method for synthesizing oxidized coenzyme Q10 from scratch, comprising: using the above-mentioned Rhodobacter sphaeroides to ferment with glucose or glycerol as a substrate to obtain oxidized coenzyme Q10.

[0012] The present invention also provides a method for synthesizing oxidized coenzyme Q10 from scratch, comprising: inoculating the above-mentioned Rhodobacter sphaeroides into a culture medium, and fermenting to obtain oxidized coenzyme Q10.

[0013] In some specific embodiments of the present invention, the above method comprises the following steps:

[0014] Step (1): inoculating the above-mentioned Rhodobacter sphaeroides into a solid culture medium, culturing, and obtaining a single colony;

[0015] Step (2): inoculating the single colony into a seed culture medium, culturing, and obtaining a seed solution;

[0016] Step (3): inoculating the seed liquid into a fermentation medium, wherein the fermentation medium contains VB1 and ferrous sulfate, wherein the concentration of the VB1 during inoculation may be 10-50 ppm, 10-45 ppm, 10-40 ppm, 10-30 ppm or 15-25 ppm, and the concentration of the ferrous sulfate may be 0.5-5 g / L, 0.5-4 g / L, 0.5-3 g / L, 1-3 g / L or 0.5-2.5 g / L, and the fermentation time may be 55-65 h, 56-64 h, 57-63 h, 58-62 h or 59-61 h;

[0017] Step (4): the residual sugar concentration is set to 2.5-7.5 g / L, 3-7 g / L, 3.5-6.5 g / L, 4-6 g / L or 4.5-5.5 g / L, the residual phosphorus concentration is set to 10-30 ppm, 12-28 ppm, 14-26 ppm, 16-24 ppm, 18-22 ppm or 19-21 ppm, and the fermentation is continued for 55-65 h, 56-64 h, 57-63 h, 58-62 h or 59-61 h to obtain oxidized coenzyme Q10.

[0018] In some specific embodiments of the present invention, the fermentation after step (2) of the above method is divided into an early stage and a late stage;

[0019] In the early stage, the seed liquid is inoculated into the fermentation medium, the initial concentrations of VB1 and ferrous sulfate are set, and the fermentation is carried out, which may include the step of adding supplementary feed, and the fermentation time may be 55-65 h, 56-64 h, 57-63 h, 58-62 h or 59-61 h;

[0020] In the later stage, the fermentation is continued, and the residual sugar concentration and the residual phosphorus concentration are controlled. The ventilation volume and / or dissolved oxygen can also be controlled. The fermentation time can be 55-65 h, 56-64 h, 57-63 h, 58-62 h or 59-61 h.

[0021] In some specific embodiments of the present invention, the inoculation ratio of the above method is 5% to 15%, 6% to 14%, 7% to 13%, 8% to 12% or 9% to 11%.

[0022] In some specific embodiments of the present invention, the fermentation temperature of the above method is 25-35°C, 26-34°C, 27-33°C, 28-32°C, 29-31°C, 30-31°C, 30-32°C, 30-33°C, 30-34°C or 30-35°C.

[0023] In some specific embodiments of the present invention, the dissolved oxygen in the fermentation in step (3) of the above method is 30% to 40%, 31% to 39%, 32% to 38%, 33% to 37% or 34% to 36%.

[0024] In some specific embodiments of the present invention, the dissolved oxygen in the fermentation in step (4) of the above method is 5% to 30%, 3% to 25%, 4% to 20%, 5% to 15%, 6% to 14%, 7% to 13%, 8% to 12% or 9% to 11%.

[0025] In some specific embodiments of the present invention, the tank pressure of the fermentation in the above method is 0.02-0.06 MPa, 0.02-0.05 MPa, 0.02-0.04 MPa or 0.025-0.035 MPa.

[0026] In some specific embodiments of the present invention, the aeration ratio of the fermentation in the above method is 0.2-1 vvm, 0.2-0.8 vvm, 0.2-0.6 vvm, 0.2-0.4 vvm or 0.2-0.3 vvm.

[0027] In some specific embodiments of the present invention, the stirring speed in the above method is automatically adjusted according to the dissolved oxygen, and can be 100-700 rpm.

[0028] In some specific embodiments of the present invention, the concentrations of the components of the solid culture medium in the above method are as follows: yeast extract: 7.5-22.5 g / L, 10-20 g / L, 12-18 g / L or 14-16 g / L; potassium dihydrogen phosphate: 0.5-1.5 g / L, 0.7-1.3 g / L or 0.9-1.1 g / L; magnesium sulfate heptahydrate: 0.25-0.75 g / L, 0.3-0.7 g / L, 0.4-0.6 g / L or 0.45-0.55 g / L; ferrous sulfate heptahydrate: 0.05-0.15 g / L, 0.07-0.13 g / L or 0.09-0.11 g / L; sodium chloride: 1-3 g / L, 1.2-2.8 g / L, 1.4-2.6 g / L, 1.6-2.4 g / L or 1.8~2.2 g / L; manganese sulfate tetrahydrate: 0.5~1.5 ppm, 0.7~1.3 ppm or 0.9~1.1 ppm; agar: 15~25 g / L, 17~23 g / L or 19~21 g / L.

[0029] In some specific embodiments of the present invention, the seed culture medium of the above method comprises: 5-15 g / L, 7-13 g / L or 9-11 g / L glucose; 2.5-7.5 g / L, 4-6 g / L or 4.5-5.5 g / L yeast extract; 2.5-7.5 g / L, 3-7 g / L, 4-6 g / L or 4.5-5.5 g / L corn steep liquor powder; 1-3 g / L, 1.5-2.5 g / L or 1.9-2.1 g / L sodium chloride; 2.5-7.5 g / L, 3-7 g / L, 4-6 g / L or 4.5-5.5 g / L potassium dihydrogen phosphate; 0.1-0.3 g / L or 0.15-0.25 g / L magnesium sulfate heptahydrate; 0.05-0.15 g / L or 0.07-0.13 ... g / L ferrous sulfate heptahydrate; 0.5~1.5 ppm, 0.7~1.3 ppm or 0.9~1.1 ppm manganese sulfate tetrahydrate; 0.5~1.5 ppm, 0.7~1.3 ppm or 0.9~1.1 ppm biotin; 0.5~1.5 ppm, 0.7~1.3 ppm or 0.9~1.1 ppm niacin; 0.5~1.5 ppm, 0.7~1.3 ppm or 0.9~1.1 ppm thiamine.

[0030] In some specific embodiments of the present invention, the fermentation medium of the above method comprises: 6-18 g / L, 8-16 g / L, 10-14 g / L or 11-13 g / L glucose; 1.5-4.5 g / L, 2-4 g / L, 2.5-3.5 g / L or 2.8-3.2 g / L ammonium sulfate; 2.5-7.5 g / L, 3-7 g / L, 4-6 g / L or 4.8-5.2 g / L potassium dihydrogen phosphate; 0.5-1.5 g / L, 0.7-0.13 g / L or 0.9-0.11 g / L magnesium sulfate heptahydrate; 0.5-5 g / L, 0.7-4.8 g / L, 0.9-4.6 g / L, 1.1-4.4 g / L, 1.3-4.2 g / L, 1.9-2.1 g / L or 3.6-4.1 g / L ferrous sulfate heptahydrate; 1~3 g / L or 1.5~2.5 g / L sodium chloride; 4~12 g / L, 6~10 g / L or 7~9 g / L corn steep liquor powder; 1.5~4.5 g / L, 2~4 g / L or 2.5~3.5 g / L glutamic acid; 25~75 ppm, 30~70 ppm, 35~65 ppm, 40~60 ppm or 45~55 ppm manganese sulfate tetrahydrate; 50~150 ppm, 75~125 ppm, 90~110 ppm or 95~105 ppm calcium chloride dihydrate; 10~50 ppm, 10~40 ppm or 15~25 ppm VB1; 0.5~1.5 ppm, 0.7~1.3 ppm or 0.9~1.1 ppm VB2; 0.5~1.5 ppm, 0.7~1.3 ppm or 0.9~1.1 ppm niacin; 0.25~0.75 ppm, 0.3~0.7 ppm, 0.4~0.6 ppm or 0.45~0.55 ppm biotin; 0.5~1.5 ppm, 0.7~1.3 ppm or 0.9~1.1 ppm folic acid.

[0031] In some specific embodiments of the present invention, the fermentation process in step (3) of the above method further includes the step of adding feed.

[0032] In some specific embodiments of the present invention, the feeding in the above method includes a primary feeding and a secondary feeding;

[0033] The feed added once includes 300-900 g / L, 400-800 g / L, 500-700 g / L or 550-650 g / L glucose, 10-30 g / L, 15-25 g / L or 18-22 g / L magnesium sulfate heptahydrate;

[0034] The secondary supplementary feed includes 125-375 g / L potassium dihydrogen phosphate.

[0035] In some specific embodiments of the present invention, during the fermentation process of step (3) and step (4) of the above method, concentrated ammonia water is used to adjust the pH to between 6.7 and 7.1, 6.75 and 7.05, 6.8 and 7.0, or 6.85 and 6.95.

[0036] The present invention has the following beneficial effects.

[0037] The strain INGIA-NM002 of the present invention can synthesize oxidized coenzyme Q10 from scratch based on glucose or glycerol, and the yield can reach 5370 mg / L, which is significantly higher than 3370 mg / L of the prior art (ATCC49419), thereby improving the yield and efficiency of industrial production. In addition, as the number of subcultures increases, the rate of decrease of the coenzyme Q10 content of the strain INGIA-NM002 of the present invention is significantly lower than that of the prior art (ATCC49419), indicating that the strain INGIA-NM002 of the present invention has a high subculture stability.

[0038] Biological Deposit Description

[0039] Biological material: INGIA-NM002, classification name: Rhodobacter sphaeroides, deposited on November 27, 2024 in the General Microbiology Center of China Culture Collection Administration. The address of the collection center is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the collection number is CGMCC No.32823. DETAILED DESCRIPTION

[0040] The present invention discloses a spherical red bacterium and its application in the de novo synthesis of oxidized coenzyme Q10. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0041] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0042] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0043] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the application is still operable. In addition, two or more steps or actions can be performed simultaneously.

[0044] The use of any and all examples or exemplary language such as "for example" or "including" herein is intended only to better illustrate the present application and is not intended to limit the scope of the present application. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the present application.

[0045] In addition, the numerical ranges and parameters used to define the present application are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally refers to the actual value within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0046] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0047] The present invention will be further described below in conjunction with the embodiments.

[0048] Example 1: Culture medium and detection method

[0049] 1. Basic information of culture medium

[0050] The information of the solid culture medium, seed culture medium, fermentation basal medium, and feed medium used in this application is as follows.

[0051] 1. Solid culture medium

[0052] Table 1: Solid culture medium components

[0053]

[0054] 2. Seed culture medium

[0055] Table 2: Seed medium components

[0056]

[0057] 3. Fermentation medium

[0058] Table 3: Fermentation basal medium components

[0059]

[0060] Table 4: Auxiliary liquid (calculated according to the initial fermentation volume, added after filtering and sterilization separately)

[0061]

[0062] 4. Feed medium

[0063] Table 5: Feed 1 (Carbon Source)

[0064]

[0065] Table 6: Feed 2 (Phosphorus source)

[0066]

[0067] Feed 3 is concentrated ammonia water, which is used to control pH.

[0068] 2. Related detection methods

[0069] 1. Wet cell content

[0070] Take 10~20 mL of fermentation liquid (volume V), add 2 mol / L hydrochloric acid to adjust the pH to 3.5~4.0, mix well, put it in a centrifuge tube (empty weight W0), and heat it in a 100℃ water bath for 10 min. After cooling, centrifuge at 10000 rpm for 5 min, discard the supernatant, and weigh W1. Calculate the wet bacterial content according to the following formula:

[0071] Wet weight (g / L) = (W1-W0) / V×1000.

[0072] 2. Residual glucose

[0073] The results were determined using SBA-2 biosensor.

[0074] 3. Solubilized Phosphate Determination

[0075] Standard curve calibration: accurately weigh 0.4394 g of sodium dihydrogen phosphate standard dried to constant weight at 105°C into a 500 mL volumetric flask and dissolve in water. Add 20 mL of 0.05 mol / L sulfuric acid test solution, shake well, dilute to scale with water to obtain a standard stock solution with a phosphorus concentration of 200 μg / mL. Accurately measure 5.0 mL of the standard stock solution into a 100 mL volumetric flask and dilute to scale with water to obtain a standard solution with a phosphorus concentration of 10 μg / mL. Accurately measure 0.5, 1.5, 2.5, 3.5, and 4.5 mL into 10 mL colorimetric tubes, add 1 mL of phosphorus test solution A and phosphorus test solution B, add water to 10 mL, mix well, heat in a boiling water bath for 30 min, cool to room temperature, add water to 25 mL and mix well, measure its absorbance at a wavelength of 650 nm, and perform a blank correction test at the same time. Draw a phosphorus standard curve based on phosphorus concentration and absorbance.

[0076] Sample dissolved phosphorus determination: Pipette 5 mL of sample into a 100 mL volumetric flask, add about 50 mL of water, and shake evenly. Add 10 mL of 10% trichloroacetic acid, shake well, let stand for 10 min, dilute to 100 mL with water, and filter. Take 2 mL of the filtrate into a 25 mL colorimetric tube, add 1 mL each of phosphorus test solution A and phosphorus test solution B, and add water to 10 mL. Heat in a boiling water bath for 30 minutes, take out, cool to room temperature, dilute to 25 mL with water, and measure the absorbance at a wavelength of 650 nm. The dissolved phosphorus calculation formula is Y= (a×A650+b), where a and b are the slope and intercept of the standard curve, respectively.

[0077] 4. Yield determination:

[0078] (1) Chromatographic conditions

[0079] Chromatographic column: C18, 4.6×150 mm, 5 μm; detection wavelength: 275 nm; flow rate: 1.5 mL / min; column temperature: 35℃; injection volume: 20 μL; mobile phase A: anhydrous methanol, mobile phase B: anhydrous ethanol, ratio: 65:35.

[0080] (2) Preparation of standard solution

[0081] Take 20 mg of coenzyme Q10 standard and place it in a 100 mL brown volumetric flask. Add 1 mL of n-hexane to dissolve it, dilute it to the mark with ethanol, and shake well to use as the standard solution.

[0082] (3) Calculation of standard solution concentration

[0083] The calculation formula is C s (mg / L) = W × P × 1000 / 100;

[0084] Wherein, W is the weight of the coenzyme Q10 working reference substance, mg; P is the purity of the coenzyme Q10 working reference substance, %.

[0085] (4) Sample processing

[0086] Take 5 mL of fermentation broth, place it in a 50 mL brown volumetric flask, add 1 drop of hydrochloric acid (6 mol / L), and shake gently. Add 10 mL of acetone, shake gently, then add about 2 mL of 30% hydrogen peroxide (total volume 4%), and shake gently. Add 30 mL of anhydrous ethanol, ultrasonically extract for 45 min (water temperature 55°C), take out, cool, add anhydrous ethanol to dilute to the scale, and shake well. Filter with a 0.22 μm filter head, discard the initial filtrate, and take the subsequent filtrate as the test solution.

[0087] (5) Sample measurement

[0088] Take 20 μL of the reference solution and the test solution, respectively, and calculate the content of the sample by the external standard method based on the peak area of ​​reduced Q10 (RRT about 0.6) and oxidized Q10.

[0089] The calculation formula is C = (A1 + A2 × 7.5) × Cs × N / As / (V or W);

[0090] Wherein, C: unit content, mg / L or mg / g; A1: oxidized peak area; A2: reduced peak area; As: reference peak area; Cs: reference solution concentration, mg / L; V: sample volume, mL, or weight, g; N: sample dilution multiple.

[0091] Example 2: Fermentation method for producing oxidized coenzyme Q10

[0092] 1. Effects of ferrous sulfate concentration, VB1 concentration, late residual sugar concentration, and late residual phosphorus concentration on fermentation yield

[0093] The initial dosage of VB1 and ferrous sulfate, and the residual concentrations of phosphorus and sugar in the later stage were combined and optimized, and different results were obtained in terms of bacterial content and yield in the tank.

[0094] (1) Activation of strains: Take out the glycerol tube containing the INGIA-NM002 strain (CGMCC No. 32823), streak it on a solid culture medium plate, and culture it at 25-35°C for 3-7 days;

[0095] (2) Seed culture: Pick the single colonies activated in step (1) and inoculate them into the seed culture medium. Each 500 mL shake flask is filled with 100 mL of liquid. Pick 3 to 5 single colonies and culture them at 200 rpm and 30 to 35°C for 24 to 30 h to obtain the seed solution.

[0096] (3) Fermentation culture: The seed liquid obtained in step (2) above was inoculated into the fermentation basal medium, with an inoculation amount of 10%. The initial culture conditions were a tank pressure of 0.03 MPa, a rotation speed of 100 rpm, a ventilation ratio of 0.4 vvm, a tank temperature of 34°C, an initial sugar concentration of 12 g / L, a phosphorus concentration of 1.14 g / L, and ferrous sulfate concentration and VB1 concentration were set according to Table 8. Feed 2 was added after about 4 to 5 hours of fermentation. After 6 to 8 hours of fermentation, the sugar was exhausted and the pH rose, and feed 1 was added. During this period, samples were taken every 4 hours to measure sugar and phosphorus. The sugar concentration was controlled at 10 g / L and phosphorus at 500 ppm in the first 60 hours. From 60 hours to the end of fermentation, the late residual sugar concentration and late residual phosphorus concentration were controlled according to the parameters in Table 8, and the late dissolved oxygen was controlled at 5% to 30%. The pH value of the fermentation liquid was adjusted between 6.7 and 7.1 by adding ammonia water throughout the process. The fermentation was terminated when the bacterial staining became lighter, part of the mycelium was autolyzed, and the yield increased slowly. Among them, the 60 hours are used as the boundary to distinguish between the "early stage" and the "late stage".

[0097] The fermentation environment condition parameters are summarized in Table 7, and the optimized settings of individual key parameter combinations are shown in Table 8.

[0098] Table 7: Summary of fermentation environmental condition parameters

[0099]

[0100] Table 8: Optimization settings for individual key parameter combinations

[0101]

[0102] After the fermentation, the tank was released and the coenzyme Q10 production (mg / L) was determined by HPLC. Chromatographic conditions: chromatographic column: C18, 4.6×150 mm, 5 μm; detection wavelength: 275 nm; flow rate: 1.5 mL / min; column temperature: 35°C; injection volume: 20 μL; mobile phase A: anhydrous methanol, phase B: anhydrous ethanol, ratio: 65:35.

[0103] The bacterial content and yield results are shown in Table 9.

[0104] Table 9: Bacterial content and yield

[0105]

[0106] It can be seen that the production of oxidized coenzyme Q10 is highest when the ferrous sulfate concentration is 2 g / L, the VB1 concentration is 20 ppm, the late residual sugar concentration is 5 g / L, and the late residual phosphorus concentration is 20 ppm.

[0107] 2. Effect of dissolved oxygen level in the late stage on fermentation yield

[0108] The fermentation method was the same as above, with the concentration of ferrous sulfate being 2 g / L, the concentration of VB1 being 20 ppm, the concentration of residual sugar in the later stage being 5 g / L, and the concentration of residual phosphorus in the later stage being 20 ppm. The dissolved oxygen level after 60 h was investigated to obtain different fermentation results, and the results are shown in Table 10.

[0109] Table 10: Effect of dissolved oxygen level in the late stage on fermentation yield

[0110]

[0111] It can be seen that the production of oxidized coenzyme Q10 is highest when the ventilation ratio is 0.2 vvm and the dissolved oxygen is controlled at 10% in the late stage.

[0112] 3. Effect of strains on fermentation yield (comparative ratio)

[0113] The commercially available Rhodobacter sphaeroides (Scientific name: Rhodobacter sphaeroides) with the number ATCC49419 was used, and the fermentation method was consistent with the optimal fermentation method of the above-mentioned preserved strain. The results are shown in Table 11.

[0114] Table 11: ATCC49419 yield

[0115]

[0116] It can be seen that compared with ATCC49419, the yield of the strain INGIA-NM002 of the present invention can reach 5370 mg / L.

[0117] Example 3: Continuous passage verification

[0118] Experimental methods:

[0119] The strain INGIA-NM002 of the present invention and the aforementioned strain ATCC49419 were inoculated on a plate culture medium for cultivation. After the colonies matured, several single colonies that met the characteristics (full, convex, and uniform in size) were selected and transferred into a test tube containing 9 mL of sterile water. After being shaken and mixed by a vortex oscillator, multiple dilutions were performed. 5 ~10 6 The bacterial suspension was inoculated into plates, 100-150 μL of the bacterial suspension was added to each plate, and the plate was evenly spread with a sterilized applicator. The above steps were followed for 5 consecutive subcultures. After each subculture, the fermentation was carried out according to the above fermentation process, the coenzyme Q10 content was detected, and the colony morphology was observed to evaluate the subculture stability. The results are shown in Tables 12 and 13.

[0120] Table 12: Continuous passage verification results of strain INGIA-NM002

[0121]

[0122] Table 13: Continuous passage verification results of strain ATCC49419

[0123]

[0124] It can be seen from Tables 12 and 13 that as the number of subcultures increases, the rate of decrease in the coenzyme Q10 content of the strain INGIA-NM002 of the present invention is significantly lower than that of the prior art (ATCC49419), which shows that the strain INGIA-NM002 of the present invention has a higher subculture stability.

[0125] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Rhodobacterium sphaeroides ( Rhodobacter sphaeroides ) INGIA-NM002, characterized in that, The deposit number is CGMCC No. 32823.

2. The spherical red bacteria as claimed in claim 1 ( Rhodobacter sphaeroides ) Application of INGIA-NM002 in the de novo synthesis of oxidized coenzyme Q10.

3. A method for synthesizing oxidized coenzyme Q10 from scratch, characterized in that: include: Using the spherical red bacteria described in claim 1 ( Rhodobacter sphaeroides INGIA-NM002 is fermented with glucose or glycerol as substrate to obtain oxidized coenzyme Q10.

4. The method according to claim 3, characterized in that The following steps are involved: Step (1): the spherical red bacteria described in claim 1 ( Rhodobacter sphaeroides ) INGIA-NM002 was inoculated into solid culture medium and cultured to obtain a single colony; Step (2): inoculating the single colony into a seed culture medium, culturing, and obtaining a seed solution; Step (3): inoculating the seed liquid into a fermentation medium, wherein the fermentation medium contains VB1 and ferrous sulfate, wherein the concentration of the VB1 during inoculation is 10-30 ppm, and the concentration of the ferrous sulfate is 1-3 g / L, and fermenting for 55-65 h; Step (4): The residual sugar concentration is set to 2.5-7.5 g / L, the residual phosphorus concentration is set to 10-30 ppm, and the fermentation is continued for 55-65 hours to obtain oxidized coenzyme Q10.

5. The method according to claim 4, characterized in that The preparation concentration of each component of the solid culture medium is: yeast extract 7.5-22.5 g / L, potassium dihydrogen phosphate 0.5-1.5 g / L, magnesium sulfate heptahydrate 0.25-0.75 g / L, ferrous sulfate heptahydrate 0.05-0.15 g / L, sodium chloride 1-3 g / L, manganese sulfate tetrahydrate 0.5-1.5 ppm, agar 15-25 g / L.

6. The method according to claim 4, characterized in that The seed culture medium comprises 5-15 g / L glucose, 2.5-7.5 g / L yeast extract, 2.5-7.5 g / L corn steep liquor dry powder, 1-3 g / L sodium chloride, 2.5-7.5 g / L potassium dihydrogen phosphate, 0.1-0.3 g / L magnesium sulfate heptahydrate, 0.05-0.15 g / L ferrous sulfate heptahydrate, 0.5-1.5 ppm manganese sulfate tetrahydrate, 0.5-1.5 ppm biotin, 0.5-1.5 ppm nicotinic acid, and 0.5-1.5 ppm thiamine.

7. The method according to claim 4, characterized in that The fermentation medium comprises 6-18 g / L glucose, 1.5-4.5 g / L ammonium sulfate, 2.5-7.5 g / L potassium dihydrogen phosphate, 0.5-1.5 g / L magnesium sulfate heptahydrate, 0.5-5 g / L ferrous sulfate heptahydrate, 1-3 g / L sodium chloride, 4-12 g / L corn steep liquor dry powder, 1.5-4.5 g / L glutamic acid, 25-75 ppm manganese sulfate tetrahydrate, 50-150 ppm calcium chloride dihydrate, 10-30 ppm VB1, 0.5-1.5 ppm VB2, 0.5-1.5 ppm nicotinic acid, 0.25-0.75 ppm biotin, and 0.5-1.5 ppm folic acid.

8. The method according to claim 4, characterized in that The fermentation process in step (3) also includes the step of adding feed.

9. The method according to claim 8, characterized in that The feeding includes primary feeding and secondary feeding; The one-time supplementary feed includes 300-900 g / L glucose and 10-30 g / L magnesium sulfate heptahydrate; The secondary supplementary feed includes 125-375 g / L potassium dihydrogen phosphate.

10. The method according to claim 8, characterized in that The fermentation in step (4) has an aeration ratio of 0.2-1 vvm and a dissolved oxygen of 2%-30%.

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