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

Abstract

The present invention relates to the field of biotechnology, and particularly to a Rhodobacter sphaeroides and its application in de novo synthesis of oxidized coenzyme Q10. The present invention provides Rhodobacter sphaeroides ( Rhodobacter sphaeroides ) INGIA-NM002, with the preservation number of CGMCC No. 32823. Using this strain to ferment and produce oxidized coenzyme Q10 with substrates (glucose or glycerol) can improve the yield and efficiency of industrial production to meet market demands.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a Rhodobacter sphaeroides and its application in de novo synthesis of oxidized coenzyme Q10. Background Art

[0002] Coenzyme Q10 (CoQ10), with the chemical name of 2,3-dimethoxy-5-methyl-6-decaprenylbenzoquinone, is widely distributed on the biological membranes of various organisms and is an important electron acceptor in the cell respiratory chain. Coenzyme Q10 is a natural antioxidant and cell metabolism activator, which can prevent and treat various diseases, and at the same time has a wide range of applications in combating fatigue, treating heart failure, delaying skin aging, etc., resulting in a further expansion of domestic and foreign demand.

[0003] At present, CoQ10 can be prepared by three methods: natural extraction, chemical synthesis and biosynthesis. Among them, the natural extraction method is restricted by raw materials and sources. The content of CoQ10 in animals and plants is low, and large-scale production is limited to a certain extent. The cost of the products produced is high and the price is expensive. The products of the chemical synthesis method are a mixture of cis-trans isomers and have low biological activity. The CoQ10 produced by biosynthesis, that is, fermentation method, has low cost, no optical isomers, high biological activity, and good application effect in large-scale production. Therefore, biosynthesis is the mainstream production process in industrial production.

[0004] There have been many research reports on the fermentation processes for preparing CoQ10 by fermenting Aspergillus fumigatus, Rhodopseudomonas capsulata, Agrobacterium tumefaciens, Rhodobacter sphaeroides, etc. At present, the strains used for fermentative production of CoQ10 still need to be further improved to improve the efficiency of industrial production and meet the market demand. Summary of the Invention

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

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

[0007] The present invention provides a Rhodobacter sphaeroides with the preservation number of CGMCC No. 32823.

[0008] The present invention also provides the application of the above-mentioned Rhodobacter sphaeroides in the synthesis of 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 de novo synthesizing oxidized coenzyme Q10, which includes: fermenting the above-mentioned Rhodobacter sphaeroides using glucose or glycerol as a substrate to obtain oxidized coenzyme Q10.

[0012] The present invention also provides a method for de novo synthesizing oxidized coenzyme Q10, which includes: 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 includes the following steps:

[0014] Step (1): Inoculate the above-mentioned Rhodobacter sphaeroides into a solid culture medium, culture to obtain single colonies;

[0015] Step (2): Inoculate the single colonies into a seed culture medium, culture to obtain a seed solution;

[0016] Step (3): Inoculate the seed solution into a fermentation culture medium, the fermentation culture medium contains VB1 and ferrous sulfate. When inoculating, the concentration of VB1 can be 10 - 50 ppm, 10 - 45 ppm, 10 - 40 ppm, 10 - 30 ppm or 15 - 25 ppm, and the concentration of ferrous sulfate can 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 can be 55 - 65 h, 56 - 64 h, 57 - 63 h, 58 - 62 h or 59 - 61 h;

[0017] Step (4): Make the residual sugar concentration be 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, make the residual phosphorus concentration be 10 - 30 ppm, 12 - 28 ppm, 14 - 26 ppm, 16 - 24 ppm, 18 - 22 ppm or 19 - 21 ppm, and continue to ferment 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 a prophase and a anaphase;

[0019] In the early stage, inoculate the seed liquid into the fermentation medium, set the initial concentrations of VB1 and ferrous sulfate and ferment, which may include the step of adding supplementary feed. 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, continue fermentation and control the residual sugar concentration and residual phosphorus concentration. It is also possible to control the ventilation volume and / or dissolved oxygen. The fermentation time may 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 - mentioned method is 5% - 15%, 6% - 14%, 7% - 13%, 8% - 12%, or 9% - 11%.

[0022] In some specific embodiments of the present invention, the fermentation temperature of the above - mentioned 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 during the fermentation in step (3) of the above - mentioned method is 30% - 40%, 31% - 39%, 32% - 38%, 33% - 37%, or 34% - 36%.

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

[0025] In some specific embodiments of the present invention, the tank pressure during the fermentation of the above - mentioned 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 ventilation ratio during the fermentation of the above - mentioned 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 during the fermentation of the above - mentioned method is automatically adjusted according to the dissolved oxygen and may be 100 - 700 rpm.

[0028] In some specific embodiments of the present invention, the preparation concentrations of the components of the solid 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 medium in the above method comprises: glucose: 5 - 15 g / L, 7 - 13 g / L, or 9 - 11 g / L; yeast extract: 2.5 - 7.5 g / L, 4 - 6 g / L, or 4.5 - 5.5 g / L; dry corn steep liquor: 2.5 - 7.5 g / L, 3 - 7 g / L, 4 - 6 g / L, or 4.5 - 5.5 g / L; sodium chloride: 1 - 3 g / L, 1.5 - 2.5 g / L, or 1.9 - 2.1 g / L; potassium dihydrogen phosphate: 2.5 - 7.5 g / L, 3 - 7 g / L, 4 - 6 g / L, or 4.5 - 5.5 g / L; magnesium sulfate heptahydrate: 0.1 - 0.3 g / L or 0.15 - 0.25 g / L; ferrous sulfate heptahydrate: 0.05 - 0.15 g / L or 0.07 - 0.13 g / L; 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; nicotinic acid: 0.5 - 1.5 ppm, 0.7 - 1.3 ppm, or 0.9 - 1.1 ppm; thiamine: 0.5 - 1.5 ppm, 0.7 - 1.3 ppm, or 0.9 - 1.1 ppm.

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

[0031] In some specific embodiments of the present invention, during the fermentation in step (3) of the above method, there is also a step of adding supplementary feed.

[0032] In some specific embodiments of the present invention, the supplementary feed of the above method includes a first addition and a second addition;

[0033] The supplementary feed for the first addition comprises glucose at 300 - 900 g / L, 400 - 800 g / L, 500 - 700 g / L or 550 - 650 g / L, and magnesium sulfate heptahydrate at 10 - 30 g / L, 15 - 25 g / L or 18 - 22 g / L;

[0034] The supplementary feed for the second addition comprises potassium dihydrogen phosphate at 125 - 375 g / L.

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

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

[0037] The strain INGIA - NM002 of the present invention can de novo synthesize oxidized coenzyme Q10 based on glucose or glycerol, with a yield of up to 5370 mg / L, significantly higher than 3370 mg / L of the prior art (ATCC49419), improving the yield and efficiency of industrial production. In addition, as the number of passages 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), indicating that the strain INGIA - NM002 of the present invention has relatively high passage stability.

[0038] Biological deposit description

[0039] Biological material: INGIA - NM002, classified as Rhodobacter sphaeroides, was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on November 27, 2024. The address of the deposit center is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC No. 32823. Detailed implementation manners

[0040] The present invention discloses a Rhodobacter sphaeroides and its application in de novo synthesizing oxidized coenzyme Q10. Those skilled in the art can draw on the content of this article and appropriately modify 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 methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate alterations and combinations to 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..." individually includes each of the objects recited after the expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

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

[0043] It should be understood that as long as the present application remains operable, the order of steps or the order of performing certain actions is not important. In addition, two or more steps or actions can be carried out simultaneously.

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

[0045] In addition, the numerical ranges and parameters used to define the present application are approximate values. The relevant values in the specific embodiments have been presented as precisely as possible herein. However, any value inherently and inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise clearly stated, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified by "about". Herein, "about" generally means that the actual value is 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 ordinary commercially available products and can be purchased from the market.

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

[0048] Example 1: Culture Medium and Detection Method

[0049] I. Basic Information of the Culture Medium

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

[0051] 1. Solid Culture Medium

[0052] Table 1: Components of the Solid Culture Medium

[0053]

[0054] 2. Seed Culture Medium

[0055] Table 2: Components of the Seed Culture Medium

[0056]

[0057] 3. Fermentation Culture Medium

[0058] Table 3: Components of the basic fermentation medium

[0059]

[0060] Table 4: Supplementary liquid (filtered and sterilized separately and added according to the initial fermentation volume)

[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 for pH control.

[0068] II. Related detection methods

[0069] 1. Wet cell content

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

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

[0072] 2. Glucose residue

[0073] Determined using an SBA-2 type biosensor.

[0074] 3. Dissolved phosphorus determination

[0075] Standard curve verification: Accurately weigh 0.4394 g of sodium dihydrogen phosphate reference standard dried to constant weight at 105 °C into a 500 mL volumetric flask, dissolve it with water. Add 20 mL of 0.05 mol / L sulfuric acid test solution, shake well, and dilute to the mark 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, dilute to the mark 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 respectively into 10 mL colorimetric tubes, add 1 mL of phosphorus test solution A and phosphorus test solution B respectively, add water to 10 mL, mix well, heat in a boiling water bath for 30 min, cool to room temperature, make up the volume to 25 mL and mix well, measure the absorbance at a wavelength of 650 nm, and at the same time perform a blank correction test. Draw the phosphorus standard curve based on the phosphorus concentration and absorbance.

[0076] Determination of soluble phosphorus in samples: Pipette 5 mL of the sample into a 100 mL volumetric flask, add about 50 mL of water, and shake well. 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 subsequent 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 formula for calculating soluble phosphorus 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 °C; Injection volume: 20 μL; Mobile phase A: anhydrous methanol, B: anhydrous ethanol, ratio: 65:35.

[0080] (2)Preparation of reference standard solution

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

[0082] (3)Calculation of the concentration of the reference standard solution

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

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

[0085] (4) Sample treatment

[0086] Take 5 mL of the 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 (4% of the total volume), and shake gently. Add 30 mL of absolute ethanol, extract ultrasonically for 45 min (water temperature 55 °C), take it out, cool it, and dilute it to the scale with absolute ethanol, then 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 determination

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

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

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

[0091] Example 2: Method for fermenting oxidized coenzyme Q10

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

[0093] Combine and optimize the initial dosages of VB1 and ferrous sulfate, and the residual concentrations of phosphorus and sugar in the later stage, and different results of the bacterial content and yield at the end of fermentation are obtained.

[0094] (1) Strain activation: Take out the glycerol tube of the preserved INGIA-NM002 strain (preservation number: CGMCC No. 32823) and streak it on a solid 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 medium. Each 500 mL shake flask is filled with 100 mL of liquid. Pick 3 - 5 single colonies and culture them at 200 rpm and 30 - 35 °C for 24 - 30 h to obtain the seed liquid;

[0096] (3)Fermentation culture: The seed liquid obtained in the above step (2) was inoculated into the basic fermentation 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, an aeration 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 the ferrous sulfate concentration and VB1 concentration were set according to Table 8; about 4 - 5 h after fermentation, feed 2 was added, the sugar was exhausted after 6 - 8 h of fermentation, the pH increased, and then feed 1 was added. Samples were taken every 4 h to measure sugar and phosphorus. The sugar concentration was controlled at 10 g / L and the phosphorus at 500 ppm in the first 60 h. After 60 h until the end of fermentation, the residual sugar concentration and residual phosphorus concentration in the later stage were controlled according to the parameters in Table 8, and the dissolved oxygen in the later stage was controlled between 5% - 30%. The pH value of the fermentation broth was adjusted to be between 6.7 - 7.1 by adding ammonia water throughout the process. When the color of the bacterial stain became lighter, some hyphae autolyzed, and the yield growth slowed down, the fermentation was terminated. Among them, the "early stage" and "later stage" were distinguished with 60 h as the boundary.

[0097] The summary of fermentation environmental condition parameters is shown 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: Optimized settings of individual key parameter combinations

[0101]

[0102] After the fermentation ended, the tank was emptied, and the yield of coenzyme Q10 (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, B: anhydrous ethanol, ratio: 65:35.

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

[0104] Table 9: Bacterial content and yield

[0105]

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

[0107] II. Influence of dissolved oxygen level in the later stage on fermentation yield

[0108] The fermentation method is the same as above, where the concentration of ferrous sulfate is 2 g / L, the concentration of VB1 is 20 ppm, the residual sugar concentration in the later stage is 5 g / L, and the residual phosphorus concentration in the later stage is 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 later stage on fermentation yield

[0110]

[0111] It can be seen from this that when the aeration ratio in the later stage is 0.2 vvm and the dissolved oxygen in the later stage is controlled at 10%, the yield of coenzyme Q10 is the highest.

[0112] III. Effect of strains on fermentation yield (comparative example)

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

[0114] Table 11: Yield of ATCC49419

[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: Verification of continuous passage

[0118] Experimental method:

[0119] The strain INGIA-NM002 of the present invention and the aforementioned strain ATCC49419 were inoculated on a plate medium for cultivation. After the colonies matured, several single colonies meeting the characteristics (plump, convex, and uniform in size) were selected and transferred into a test tube containing 9 mL of sterile water. After being shaken and mixed evenly by a vortex oscillator, serial dilutions were performed. Take the bacterial suspension with a dilution factor of 10 5 ~10 6 and inoculate it on a plate. Add 100 - 150 μL of the bacterial suspension to each plate and spread it evenly with a sterilized spreader. Continuously passage 5 times according to the above steps. After each passage, ferment according to the above fermentation process, detect the content of coenzyme Q10, and observe the colony morphology to evaluate the passage stability. The results are shown in Tables 12 and 13.

[0120] Table 12: Results of continuous passage verification of strain INGIA-NM002

[0121]

[0122] Table 13: Verification results of successive subcultures of strain ATCC49419

[0123]

[0124] It can be seen from Table 12 and Table 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). It can be seen that the subculture stability of the strain INGIA-NM002 of the present invention is relatively high.

[0125] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

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

2. Use of Rhodobacter sphaeroides INGIA-NM002 as claimed in claim 1 in the de novo synthesis of oxidized coenzyme Q10.

3. A method for synthesizing oxidized coenzyme Q10 from scratch, characterized in that: include: The Rhodobacter sphaeroides INGIA-NM002 described in claim 1 is used for fermentation with glucose or glycerol as a substrate to obtain oxidized coenzyme Q10.

4. The method according to claim 3, characterized in that The following steps are involved: Step (1): inoculating the Rhodobacter sphaeroides INGIA-NM002 described in claim 1 into a solid culture medium, culturing, and obtaining 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 heptahydrate, wherein the concentration of the VB1 during inoculation is 10-30 ppm, and the concentration of the ferrous sulfate heptahydrate 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 125-375 g / L potassium dihydrogen phosphate; The secondary supplementary feed includes 300-900 g / L glucose and 10-30 g / L magnesium sulfate heptahydrate.

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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