Method for de novo synthesis of reduced coenzyme Q10

Abstract

The invention relates to the technical field of biology, in particular to a method for de novo synthesis of reduced coenzyme Q10. The method provided by the invention is based on coenzyme Q10 reductases from five sources and takes oxidized coenzyme Q10 as a substrate for preparation, the oxidized coenzyme Q10 can be synthesized from Rhodobacter sphaeroides INGIA-NM002 through special condition parameters, and the whole process flow has the advantages of high yield, high purity, low cost, high safety, suitability for industrial production and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for synthesizing reduced coenzyme Q10 from scratch. Background Art

[0002] Coenzyme Q10 (CoQ10 for short) is a fat-soluble quinone compound widely distributed in human cells. It plays an important role in the mitochondrial electron transport chain of cells and participates in energy production. CoQ10 has two forms: oxidized and reduced. Reduced coenzyme Q10 (Ubiquinol) has high bioavailability and good oral absorption. It has the functions of anti-oxidation (scavenging free radicals, etc.), preventing vascular atherosclerosis, and improving chronic respiratory diseases. It has a wide range of applications in food (including beverages), nutritional products (including nutritional supplements), feed, daily chemicals (including cosmetics) and other fields.

[0003] Traditional methods for producing reduced coenzyme Q10 include synthesis, fermentation, extraction from natural products, etc. At present, the common process for producing reduced coenzyme Q10 is to use a reducing agent to reduce oxidized coenzyme Q10, such as sodium bisulfite, sodium thiosulfate, sodium borohydride, potassium borohydride, stannous chloride, lithium aluminum hydride, etc. The disadvantage of this process is that the reducing agent will affect the quality of reduced coenzyme Q10, and complex methods need to be implemented to remove them, resulting in higher costs and poorer quality. In addition, the by-product (Z)-isomer presents potential safety hazards and is difficult to remove when mixed with the final product. The content of coenzyme Q10 in animals and plants is low, so the cost of extracting reduced coenzyme Q10 from organisms is high, and large-scale production is limited.

[0004] It can be seen that the current production process may not meet the needs of commercial-scale production, and the production efficiency is low, resulting in high costs. Summary of the invention

[0005] In view of this, the present invention provides a process for synthesizing reduced coenzyme Q10 from scratch, which has the advantages of high yield, high purity, low cost, high safety, and is more suitable for industrial production.

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

[0007] The present invention provides a method for synthesizing reduced coenzyme Q10 from scratch, comprising: synthesizing oxidized coenzyme Q10, and then using the oxidized coenzyme Q10 as a substrate to synthesize reduced coenzyme Q10 based on coenzyme Q10 reductase.

[0008] In some specific embodiments of the present invention, the synthesizing oxidized coenzyme Q10 in the above method comprises: synthesizing oxidized coenzyme Q10 using Rhodobacter sphaeroides that can synthesize oxidized coenzyme Q10.

[0009] In some specific embodiments of the present invention, the method comprises: using glucose as a substrate, using Rhodobacter sphaeroides that can synthesize oxidized coenzyme Q10 to synthesize oxidized coenzyme Q10, and then using the oxidized coenzyme Q10 as a substrate, based on coenzyme Q10 reductase 1 derived from Deltaproteobacteria bacterium, coenzyme Q10 reductase 2 derived from Coprinopsis cinerea okayama7#130, coenzyme Q10 reductase 3 derived from Pseudocercosporaeumusae, coenzyme Q10 reductase 4 derived from Nannizzia gypsea CBS 118893 or coenzyme Q10 reductase 5 derived from Pestalotiopsis fici W106-1 to synthesize reduced coenzyme Q10.

[0010] In some specific embodiments of the present invention, the NCBI GenPept accession number of the coenzyme Q10 reductase 1 in the above method is MBP78919.1; the NCBI GenPept accession number of the coenzyme Q10 reductase 2 is XP_001834806.1; the NCBI GenPept accession number of the coenzyme Q10 reductase 3 is KXS94104.1; the NCBI GenPept accession number of the coenzyme Q10 reductase 4 is XP_003169128.1; and the UniProtKB accession number of the coenzyme Q10 reductase 5 is A0A067XMP1.1.

[0011] In some specific embodiments of the present invention, the method comprises: extracting oxidized coenzyme Q10 from the Rhodobacter sphaeroides, mixing (a) Tris-HCl, (b) NaCl, (c) NADH or NADPH, (d) reductase and (e) the oxidized coenzyme Q10 to obtain a reaction system, reacting the reaction system under anaerobic conditions at 40-50° C., 42.5-47.5° C. or 44-46° C. for 15-45 min, 20-40 min or 25-35 min to obtain reduced coenzyme Q10;

[0012] Alternatively, the Rhodobacter sphaeroides is cultured to obtain a fermentation broth, the fermentation broth is crushed, and then mixed with (a) Tris-HCl, (b) NaCl, (c) NADH or NADPH, and (d) reductase, and reacted at 40-50° C., 42.5-47.5° C., or 44-46° C. under anaerobic conditions for 15-45 min, 20-40 min, or 25-35 min to obtain reduced coenzyme Q10;

[0013] The reductase is the coenzyme Q10 reductase 1, the coenzyme Q10 reductase 2, the coenzyme Q10 reductase 3, the coenzyme Q10 reductase 4 or the coenzyme Q10 reductase 5.

[0014] In some specific embodiments of the present invention, in the reaction system of the above method, the concentration of Tris-HCl is 50-150 mM, 75-125 mM or 90-110 mM, the concentration of NaCl is 250-750 mM, 400-600 mM or 450-550 mM, the concentration of NADH or NADPH is 1-3 mM, 1.5-2.5 mM or 1.9-2.1 mM, and the concentration of oxidized coenzyme Q10 is 0.5-1.5 mM, 0.7-1.3 mM or 0.9-1.1 mM.

[0015] In some specific embodiments of the present invention, the volume ratio of the reductase to the reaction system in the above method is 25% to 75%, 35% to 65%, 40% to 60%, 45% to 55% or 50%;

[0016] The preparation method of the reductase comprises: synthesizing an expression vector, wherein the expression vector has a sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, introducing the expression vector into competent cells and culturing the cells to obtain a recombinant strain, culturing the recombinant strain, and waiting for the OD of the recombinant strain to reach 1. 600 When the pH value reaches 0.5-0.7 or 0.55-0.65, the bacterial solution of the recombinant strain is harvested and the bacterial cells of the recombinant strain are collected. The bacterial cells of the recombinant strain are washed, resuspended, and broken. The supernatant is taken by centrifugation to obtain the reductase.

[0017] In some specific embodiments of the present invention, the preparation method of the oxidized coenzyme Q10 in the above method comprises: centrifuging the fermentation broth of the Rhodobacter sphaeroides to obtain bacterial cells, mixing the bacterial cells with an organic solvent, stirring, standing for 40 to 60 min, 45 to 55 min or 48 to 52 min, centrifuging to obtain a supernatant organic phase, and vacuum drying the supernatant organic phase to obtain oxidized coenzyme Q10.

[0018] In some specific embodiments of the present invention, the Rhodobacter sphaeroides in the above method is Rhodobacter sphaeroides INGIA-NM002 with a deposit number of CGMCC No. 32823.

[0019] In some specific embodiments of the present invention, the method for preparing the fermentation broth of the above method comprises:

[0020] Step (1): inoculating the Rhodobacter sphaeroides INGIA-NM002 into a solid culture medium, culturing, and obtaining a single colony;

[0021] Step (2): inoculating the single colony into a seed culture medium, culturing, and obtaining a seed solution;

[0022] 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 10-30 ppm or 15-25 ppm, the concentration of the ferrous sulfate may be 1-3 g / L or 0.5-2.5 g / L, and the fermentation time may be 58-62 h or 59-61 h;

[0023] Step (4): the residual sugar concentration is set to 4-6 g / L or 4.5-5.5 g / L, the residual phosphorus concentration is set to 18-22 ppm or 19-21 ppm, and the fermentation is continued for 58-62 h or 59-61 h to obtain oxidized coenzyme Q10.

[0024] In some specific embodiments of the present invention, the fermentation temperature of the above method is 25-35°C, 30-34°C or 30-35°C.

[0025] In some specific embodiments of the present invention, the dissolved oxygen in the fermentation in step (3) of the above method is 33% to 37% or 34% to 36%.

[0026] In some specific embodiments of the present invention, the dissolved oxygen in the fermentation in step (4) of the above method is 8% to 12% or 9% to 11%.

[0027] In some specific embodiments of the present invention, the tank pressure of the fermentation in the above method is 0.02~0.04 MPa or 0.025~0.035 MPa.

[0028] In some specific embodiments of the present invention, the aeration ratio of the fermentation in the above method is 0.2-0.4 vvm or 0.2-0.3 vvm.

[0029] 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.

[0030] In some specific embodiments of the present invention, the fermentation broth of the above method may or may not include the Rhodobacter sphaeroides INGIA-NM002.

[0031] 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: 12-18 g / L or 14-16 g / L; potassium dihydrogen phosphate: 0.7-1.3 g / L or 0.9-1.1 g / L; magnesium sulfate heptahydrate: 0.4-0.6 g / L or 0.45-0.55 g / L; ferrous sulfate heptahydrate: 0.07-0.13 g / L or 0.09-0.11 g / L; sodium chloride: 1.6-2.4 g / L or 1.8-2.2 g / L; manganese sulfate tetrahydrate: 0.7-1.3 ppm or 0.9-1.1 ppm; agar: 17-23 g / L or 19-21 g / L;

[0032] The seed culture medium comprises 7-13 g / L or 9-11 g / L glucose; 4-6 g / L or 4.5-5.5 g / L yeast extract; 4-6 g / L or 4.5-5.5 g / L corn steep liquor powder; 1.5-2.5 g / L or 1.9-2.1 g / L sodium chloride; 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; 0.7-1.3 ppm or 0.9-1.1 ppm manganese sulfate tetrahydrate; 0.7-1.3 ppm or 0.9-1.1 ppm biotin; 0.7-1.3 ppm or 0.9-1.1 ppm nicotinic acid; 0.7-1.3 ppm or 0.9-1.1 ppm ppm or 0.9~1.1 ppm thiamine;

[0033] The fermentation medium comprises 10-14 g / L or 11-13 g / L glucose; 2.5-3.5 g / L or 2.8-3.2 g / L ammonium sulfate; 4-6 g / L or 4.8-5.2 g / L potassium dihydrogen phosphate; 0.7-0.13 g / L or 0.9-0.11 g / L magnesium sulfate heptahydrate; 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; 6-10 g / L or 7-9 g / L corn steep liquor powder; 2-4 g / L or 2.5-3.5 g / L glutamic acid; 40-60 ppm or 45-55 ppm manganese sulfate tetrahydrate; 90-110 ppm or 95-105 ppm calcium chloride dihydrate; 10-40 ppm or 15-25 ppm VB1; 0.7~1.3 ppm or 0.9~1.1ppm VB2; 0.7~1.3 ppm or 0.9~1.1 ppm niacin; 0.4~0.6 ppm or 0.45~0.55 ppm biotin; 0.7~1.3ppm or 0.9~1.1 ppm folic acid.

[0034] 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;

[0035] The feeding includes primary feeding and secondary feeding;

[0036] The feed added once includes 500-700 g / L or 550-650 g / L glucose, 15-25 g / L or 18-22 g / L magnesium sulfate heptahydrate;

[0037] The secondary supplementary feed includes 125-375 g / L potassium dihydrogen phosphate.

[0038] 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 6.8 and 7.0 or between 6.85 and 6.95.

[0039] The present invention has the following beneficial effects.

[0040] The experiment shows that when the coenzyme is NADH, the reduced coenzyme Q10 conversion rates of the coenzyme Q10 reductases expressed by the ecCoQH-1, ecCoQH-2, ecCoQH-3, ecCoQH-4 and ecCoQH-5 of the present invention are approximately 70.3%, 62.9%, 21.0%, 4.8% and 25.2%, respectively, and when the coenzyme is NADPH, the reduced coenzyme Q10 conversion rates of the coenzyme Q10 reductases expressed by the ecCoQH-1, ecCoQH-2, ecCoQH-3, ecCoQH-4 and ecCoQH-5 are approximately 29.0%, 16.6%, 46.1%, 29.6% and 49.4%, respectively. When the reaction temperature is 45°C, the reduced coenzyme Q10 conversion rate of the coenzyme Q10 reductase expressed by the ecCoQH-1 strain is the highest. The present invention can also obtain oxidized coenzyme Q10 based on Rhodobacter sphaeroides INGIA-NM002 through special condition parameters, and then use the oxidized coenzyme Q10 as a substrate in combination with the fermentation product of ecCoQH-1, ecCoQH-2, ecCoQH-3, ecCoQH-4 or ecCoQH-5 to achieve de novo synthesis of reduced coenzyme Q10, which has high yield, high purity, low cost, high safety, and is more suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0042] Figure 1 The results of protein electrophoresis of coenzyme Q10 reductase 1 from Deltaproteobacteria bacterium are shown;

[0043] Figure 2 The results of protein electrophoresis of coenzyme Q10 reductase 3 from Pseudocercospora eumusae are shown;

[0044] Figure 3 The protein electrophoresis results of reductase are shown, wherein, from left to right, the first, second and third lanes are coenzyme Q10 reductase 4 derived from Nannizzia gypsea CBS 118893, the fourth, fifth and sixth lanes are coenzyme Q10 reductase 5 derived from Pestalotiopsis fici W106-1, and the eighth, ninth and tenth lanes are coenzyme Q10 reductase 2 derived from Coprinopsis cinerea okayama7#130;

[0045] Figure 4The conversion rate of reduced coenzyme Q10 of different strains is shown when the coenzyme is NADH;

[0046] Figure 5 The conversion rate of reduced coenzyme Q10 of different strains is shown when the coenzyme is NADPH;

[0047] Figure 6 The reduced coenzyme Q10 conversion rate of ecCoQH-1 at different temperatures is shown.

[0048] Biological Deposit Description

[0049] 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

[0050] The present invention discloses a method for synthesizing reduced coenzyme Q10 from scratch. 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 are 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 method and application described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0051] The present application provides a method for producing reduced coenzyme Q10, wherein the method uses the following coenzyme Q10 reductase (CoQreductase) as a reductant to reduce oxidized coenzyme Q10 to generate reduced coenzyme Q10. The coenzyme Q10 reductase includes any one of the following.

[0052] Coenzyme Q10 reductase 1 from Deltaproteobacteria bacterium, NCBI GenPept accession number MBP78919.1, Escherichia coli codon-optimized nucleotide sequence:

[0053]

[0054] Coenzyme Q10 reductase 2 from Coprinopsis cinerea okayama7#130, NCBI GenPept accession number is XP_001834806.1, and the E. coli codon-optimized nucleotide sequence is:

[0055]

[0056] Coenzyme Q10 reductase 3 from Pseudocercospora eumusae, NCBI GenPept accession number is KXS94104.1, and the codon-optimized nucleotide sequence for E. coli is:

[0057]

[0058] Coenzyme Q10 reductase 4 from Nannizzia gypsea CBS 118893, NCBI GenPept accession number XP_003169128.1, E. coli codon-optimized nucleotide sequence:

[0059]

[0060] Coenzyme Q10 reductase 5 from Pestalotiopsis fici W106-1, UniProtKB / Swiss-Prot accession number is A0A067XMP1.1, and the codon-optimized nucleotide sequence of Escherichia coli is:

[0061]

[0062] As a control of the present application, the present application also relates to a reductase derived from Caldalkalibacillus thermarum, the NCBI GenPept accession number is WP_007502350.1, and the nucleotide sequence is:

[0063]

[0064] The backbone sequence of the plasmid involved in this application is:

[0065]

[0066] In this application, NADH and NADPH were purchased from MilliporeSigma.

[0067] 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.

[0068] The present invention will be further described below in conjunction with the embodiments.

[0069] Example 1: Strain construction

[0070] 1. Construction of recombinant plasmid

[0071] Construction of plasmid: The plasmid backbone is pET28a, and the sequence is shown in SEQ ID NO: 7.

[0072] According to the gene sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, plasmids pHQ-1, pHQ-2, pHQ-3, pHQ-4, and pHQ-5 were obtained by total synthesis. Among them:

[0073] pHQ-1: backbone sequence + SEQ ID NO: 1, insertion sites are NdeI and XhoI;

[0074] pHQ-2: backbone sequence + SEQ ID NO: 2, insertion sites are NdeI and XhoI;

[0075] pHQ-3: backbone sequence + SEQ ID NO: 3, insertion sites are NdeI and XhoI;

[0076] pHQ-4: backbone sequence + SEQ ID NO: 4, insertion sites are NdeI and XhoI;

[0077] pHQ-5: backbone sequence + SEQ ID NO: 5, insertion sites are NdeI and XhoI.

[0078] 2. Construction of bacteria expressing reductase

[0079] Take the construction of ecCoQH-1 expressing reductase as an example:

[0080] The above pHQ-1 plasmid (100 ng) was mixed with 100 μL of BL21 (DE3) competent cells, and after electroporation, it was transferred to LB medium and cultured for 30 min, and then evenly spread on LB plates containing the antibiotic kanamycin (50 mg / L). After overnight culture, a single colony was picked for preservation to obtain the reductase-expressing bacterium ecCoQH-1.

[0081] The construction method of the remaining reductase-expressing bacteria was the same as that of ecCoQH-1, and the reductase-expressing bacteria ecCoQH-2, ecCoQH-3, ecCoQH-4 and ecCoQH-5 were obtained respectively.

[0082] 3. Construction of control bacteria

[0083] The plasmid pHQ-ctrl used for the control bacteria was fully synthesized by the company. Among them, pHQ-ctrl is the backbone sequence + SEQID NO: 6, and the insertion sites are NdeI and XhoI.

[0084] The pHQ-ctrl plasmid (100 ng) and 100 μL of BL21 (DE3) competent cells were electroporated and transferred to LB medium for 30 min. The plates were then evenly spread on LB plates containing the antibiotic kanamycin (50 mg / L). After overnight culture, single colonies were picked for preservation to obtain the reductase-expressing bacterium ecCoQH-c.

[0085] Example 2: Enzyme activity verification and comparison

[0086] (1) Preparation of reductase by recombinant bacterial expression

[0087] The constructed ecCoQH-1, ecCoQH-2, ecCoQH-3, ecCoQH-4 and ecCoQH-5 strains were streaked onto LB plates without resistance, cultured overnight at 37°C, single clones were picked out and placed into 5 mL LB medium, and cultured at 37°C and 200 rpm until OD 600 ~0.6. Then 0.1 mM IPTG was added for induction, and the cells were cultured in a shaker at 22°C for 18 h before harvesting. The resulting bacterial solution was rinsed with saline three times, resuspended in 1 mL of saline, and then placed in an ultrasonic disruptor for full disruption. After centrifugation (12000 rpm, 1 min), the supernatant was collected to obtain coenzyme Q10 reductase 1, coenzyme Q10 reductase 2, coenzyme Q10 reductase 3, coenzyme Q10 reductase 4, and coenzyme Q10 reductase 5. The protein electrophoresis is shown in Figure 1 , Figure 2 , Figure 3 .

[0088] The ecCoQH-c strain of the control group was streaked onto LB plates without resistance, cultured overnight at 37°C, single clones were picked out and placed in 5 mL LB medium, and cultured at 37°C and 200 rpm until OD 600 ~0.6. Then 0.1 mM IPTG was added for induction, and the cells were cultured in a shaker at 22°C for 18 h before harvesting. The resulting bacterial solution was rinsed with saline three times and resuspended in 1 mL of saline. Then the cells were placed in an ultrasonic crusher for full crushing, and the supernatant was taken after centrifugation (12000 rpm, 1 min) to obtain the reductase of the control bacteria.

[0089] (2) Enzyme activity verification

[0090] ① Coenzyme is NADH

[0091] The reaction was carried out under anaerobic conditions, in which NADH was added as a coenzyme for the reaction. The final volume of the reaction mixture was 2 mL, containing 100 mM Tris-HCl (pH = 7.6), 500 mM NaCl, 2 mM NADH, 1 mM oxidized CoQ10 (purchased from Merck, purity 95%), and 1 mL of the reductase obtained above. The reaction was carried out under anaerobic conditions, i.e., in a glove box filled with nitrogen. All sample solutions were degassed under nitrogen before use, and the reaction temperature was set to 37°C.

[0092] After the reaction was carried out for 30 min, methanol was used for quenching, and then the content was quickly determined by liquid chromatography to obtain the conversion rate, such as Figure 4 Specific data are shown in Table 1.

[0093] Table 1: Conversion rate of reduced coenzyme Q10 of different strains when the coenzyme is NADH

[0094]

[0095] ② Coenzyme is NADPH

[0096] The reaction was carried out under anaerobic conditions, in which NADPH was added as a coenzyme for the reaction. The final volume of the reaction mixture was 2 mL, containing 100 mM Tris-HCl (pH = 7.6), 500 mM NaCl, 2 mM NADPH, 1 mM oxidized CoQ10, and 1 mL of the reductase obtained above. The reaction was carried out under anaerobic conditions, i.e., in a glove box filled with nitrogen. All sample solutions were degassed under nitrogen before use, and the reaction temperature was set to 37°C. After the reaction was carried out for 30 min, it was quenched with methanol, and then the content was quickly determined by liquid chromatography to obtain the conversion rate, as shown in Figure 5 The specific data are shown in Table 2.

[0097] Table 2: Conversion rate of reduced coenzyme Q10 of different strains when the coenzyme is NADPH

[0098]

[0099] HPLC detection conditions are as follows: chromatographic column: C18; mobile phase: methanol: ethanol = 1:1, flow rate: 1 mL / min, wavelength: 275 nm; sample loading: 20 μL; detection temperature: 30°C.

[0100] from Figure 4 , Figure 5 It can be seen that when the coenzyme is NADH, the conversion rate of ecCoQH-1 is the highest.

[0101] (3) Optimization of reaction temperature conditions

[0102] The ecCoQH-1 with the highest conversion rate was selected as the temperature optimization experimental strain, and its protein expression steps and reaction steps were the same as described above.

[0103] The reaction temperatures were set at 22°C, 30°C, 37°C, 45°C, 50°C, 60°C, and 70°C, respectively. The conversion results are shown in Figure 6 The specific data are shown in Table 3.

[0104] Table 3: Conversion rate of reduced coenzyme Q10 of ecCoQH-1 at different temperatures

[0105]

[0106] from Figure 6 As can be seen from Table 3, the conversion rate is highest when the reaction temperature is 45°C.

[0107] Example 3: De novo synthesis of reduced coenzyme Q10

[0108] Using glucose as a substrate, a deposited strain (INGIA-NM002, deposit number: CGMCC No. 32823) was used to ferment and produce oxidized coenzyme Q10, and ecCoQH-1 with the highest conversion rate in the aforementioned enzyme activity verification test was used to convert the oxidized coenzyme Q10 into reduced coenzyme Q10.

[0109] 1. Production of oxidized coenzyme Q10

[0110] S101. Activation of strains: Take out the glycerol tube of INGIA-NM002 strain (deposit number: CGMCC No. 32823), streak it on a solid culture medium plate, and culture it at 25-35°C for 3-7 days;

[0111] S102. Seed culture: Pick a single colony activated in step S101 and inoculate it into a seed culture medium. Specifically, pick 3 to 5 single colonies from each 500 mL shake flask containing 100 mL of liquid, and culture them at 200 rpm and 30 to 35°C for 24 to 30 h to obtain a seed solution.

[0112] S103. Fermentation culture: The seed liquid obtained in the above step S102 is inoculated into the fermentation basal medium, the inoculation amount is 10%, the initial culture conditions are tank pressure 0.03 MPa, rotation speed 100 rpm, ventilation ratio 0.4 vvm, tank temperature 34°C, initial sugar concentration 12 g / L, phosphorus concentration 1.14 g / L, ferrous sulfate concentration 2 g / L, VB1 concentration 20 ppm; after fermentation for about 4 to 5 hours, feed 2 is added, after fermentation for 6 to 8 hours, sugar is exhausted, pH rises, and feed 1 is added. During this period, samples are taken every 4 hours to measure sugar and phosphorus, and the sugar concentration is controlled at 10 g / L and phosphorus at 500 ppm in the first 60 hours. After 60 hours until the end of fermentation, the late residual sugar concentration is controlled at 5 g / L, the late residual phosphorus concentration is controlled at 20 ppm, the ventilation ratio is 0.2, and the dissolved oxygen is 10%. During the whole process, the pH value of the fermentation liquid was adjusted between 6.7 and 7.1 by adding ammonia water. When the bacterial body became lighter in color, part of the hyphae was autolyzed, and the yield increased slowly, the fermentation was terminated. The 60 h was used as the boundary to distinguish the "early stage" and the "late stage".

[0113] Chromatographic conditions for the detection of oxidized coenzyme Q10: 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.

[0114] The culture medium formula used above is as follows:

[0115] 1) Solid culture medium

[0116] Table 4: Solid culture medium components

[0117]

[0118] 2) Seed culture medium

[0119] Table 5: Seed medium components

[0120]

[0121] 3) Fermentation medium

[0122] Table 6: Fermentation basal medium components

[0123]

[0124] Table 7: Auxiliary liquid (calculated according to the initial fermentation volume, added after filtering and sterilization separately)

[0125]

[0126] 4) Feed medium

[0127] Table 8: Feed 1 (Carbon Source)

[0128]

[0129] Table 9: Feed 2 (Phosphorus source)

[0130]

[0131] Feed 3 is concentrated ammonia water, which is used to control pH.

[0132] 2. Production of Reduced Coenzyme Q10

[0133] Method 1 (using the fermentation broth of the preserved strain to purify oxidized coenzyme Q10 as a substrate):

[0134] S201. After the fermentation is completed, 10 mL of the fermentation liquid after the fermentation in step S103 is centrifuged to obtain a bacterial precipitate, 10-20 mL of an organic solvent (such as petroleum ether or ethyl acetate, petroleum ether is used in this example) is slowly added to the bacterial precipitate, stirred to fully dissolve, allowed to stand for 40-60 min, and then centrifuged at 12,000 rpm to obtain a supernatant organic phase, the organic phase is vacuum-dried at 0.1-0.5 MPa to obtain a powder, i.e., the target precursor substance (oxidized coenzyme Q10), and 10 mL of petroleum ether is added to dissolve;

[0135] S202. Take the coenzyme Q10 reductase 1 prepared above and the reductase of the control bacteria, respectively, and the target precursor substance (oxidized coenzyme Q10) in step S201, add NADH as a coenzyme, and mix thoroughly. The final volume of the reaction mixture is 2mL, including: 100 mM Tris-HCl (pH=7.6), 500 mM NaCl, 2 mM NADH, 1 mL reductase and 1 mM target precursor substance (oxidized coenzyme Q10), and the concentrations are all the final concentrations of the reaction system. The reaction is carried out under anaerobic conditions, that is, in a glove box filled with nitrogen. All sample solutions are degassed under nitrogen before use, and the reaction temperature is set to 45°C. After the reaction is carried out for 30 min, it is quenched with methanol, and then the content is quickly determined by liquid chromatography to calculate the conversion rate.

[0136] Method 2 (using the fermentation broth of the preserved strain as substrate):

[0137] S301. After the fermentation in step S103, 10 mL of the fermentation liquid was centrifuged to obtain a precipitate, which was resuspended with 2 mL of saline and then placed in an ultrasonic crusher for full crushing (stopping for one second every 4 seconds for a total of 30 minutes) to obtain a precursor fermentation liquid containing the target precursor substance (oxidized coenzyme Q10);

[0138] S302. The coenzyme Q10 reductase 1 and the control bacteria reductase prepared above are added to the precursor fermentation broth containing the target precursor substance (oxidized coenzyme Q10) in step S301, and NADH is added as a coenzyme to continue the reaction. The final volume of the reaction mixture is 2 mL, including: 100 mM Tris-HCl (pH=7.6), 500 mM NaCl, 2 mM NADH, 1 mL reductase and precursor fermentation broth (containing 1 mM oxidized coenzyme Q10, the amount added is calculated based on the HPLC quantitative result: 500 μL of the precursor fermentation broth is mixed with an equal volume of petroleum ether and ethyl acetate mixed solution, wherein the volume ratio of petroleum ether to ethyl acetate is 4:1, and after extraction, HPLC is sent for quantitative determination to obtain the concentration of oxidized coenzyme Q10 in the precursor fermentation broth, and the amount added is determined based on the concentration). The reaction is carried out under anaerobic conditions, i.e., in a glove box filled with nitrogen. All sample solutions are degassed under nitrogen before use, and the reaction temperature is set to 45°C. After the reaction was carried out for 30 min, the product was quenched with methanol, and then the content was determined by rapid liquid chromatography to calculate the conversion rate.

[0139] The specific data of the conversion rate of method 1 and method 2 are shown in Table 10.

[0140] Table 10: Conversion rate of reduced coenzyme Q10 in method 1 and method 2

[0141]

[0142] It can be seen that the oxidized coenzyme Q10 obtained by fermenting the fermentation broth obtained by the preserved strain INGIA-NM002 and then purifying it can reach a conversion rate of 71.7% under the action of the coenzyme Q10 reductase 1 of the present invention, which is significantly higher than the reductase conversion rate of the control bacteria; and it can be found that the conversion rate of 54.5% can be achieved by directly using the fermentation broth as a substrate after crushing and using the coenzyme Q10 reductase 1 of the present invention to synthesize reduced coenzyme Q10, while the reductase conversion rate of the control bacteria is only 6.8%, indicating that the coenzyme Q10 reductase of the present invention has a higher impurity tolerance and reduces the conversion cost.

[0143] 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. A method for synthesizing reduced coenzyme Q10 from scratch, characterized in that: include: Oxidized coenzyme Q10 is synthesized, and then reduced coenzyme Q10 is synthesized based on coenzyme Q10 reductase using the oxidized coenzyme Q10 as a substrate.

2. The method according to claim 1, characterized in that The coenzyme Q10 reductase is coenzyme Q10 reductase 1, coenzyme Q10 reductase 2, coenzyme Q10 reductase 3, coenzyme Q10 reductase 4 or coenzyme Q10 reductase 5; The coenzyme Q10 reductase 1 is derived from Deltaproteobacteria bacterium, and the encoding gene has at least 95% similarity to SEQ ID NO: 1; The coenzyme Q10 reductase 2 is derived from Coprinopsis cinerea okayama7# 130, encoding a gene having at least 95% similarity to SEQ ID NO: 2; The coenzyme Q10 reductase 3 is derived from Pseudocercospora eumusae, and the encoding gene has at least 95% similarity to SEQ ID NO: 3; The coenzyme Q10 reductase 4 is derived from Nannizzia gypsea CBS 118893, and the encoding gene has at least 95% similarity to SEQ ID NO: 4; The coenzyme Q10 reductase 5 is derived from Pestalotiopsis fici W106-1, and the encoding gene has at least 95% similarity to SEQ ID NO:

5.

3. The method according to claim 1 or 2, characterized in that The synthesizing oxidized coenzyme Q10 comprises: synthesizing oxidized coenzyme Q10 using Rhodobacter sphaeroides which can synthesize oxidized coenzyme Q10.

4. The method according to claim 3, characterized in that include: (A) using the Rhodobacter sphaeroides to synthesize oxidized coenzyme Q10, extracting oxidized coenzyme Q10 from the fermentation broth of the Rhodobacter sphaeroides, mixing Tris-HCl, NaCl, NADH, reductase and the oxidized coenzyme Q10 to obtain a reaction system, reacting the reaction system under anaerobic conditions at 40 to 50° C. for 15 to 45 minutes to obtain reduced coenzyme Q10; Or (B) culturing the Rhodobacter sphaeroides to obtain a fermentation broth, crushing the fermentation broth, mixing it with Tris-HCl, NaCl, NADH, and reductase, and reacting it at 40-50° C. under anaerobic conditions for 15-45 min to obtain reduced coenzyme Q10; The reductase is the coenzyme Q10 reductase 1, the coenzyme Q10 reductase 2, the coenzyme Q10 reductase 3, the coenzyme Q10 reductase 4 or the coenzyme Q10 reductase 5.

5. The method according to claim 4, characterized in that In the reaction system, the concentration of Tris-HCl is 50-150 mM, the concentration of NaCl is 250-750 mM, the concentration of NADH is 1-3 mM, and the concentration of oxidized coenzyme Q10 is 0.5-1.5 mM.

6. The method according to claim 4, characterized in that The volume ratio of the reductase to the reaction system is 25% to 75%; The preparation method of the reductase comprises: synthesizing an expression vector, wherein the expression vector has a sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, introducing the expression vector into competent cells and culturing the cells to obtain a recombinant strain, culturing the recombinant strain, and waiting for the OD of the recombinant strain to reach 1. 600 When the pH value reaches 0.5-0.7, the bacterial solution of the recombinant strain is harvested and the bacterial cells of the recombinant strain are collected. The bacterial cells of the recombinant strain are washed, resuspended, and broken. The supernatant is taken by centrifugation to obtain the reductase.

7. The method according to claim 1 or 2, characterized in that: The preparation method of the oxidized coenzyme Q10 comprises: centrifuging a fermentation broth of Rhodobacter sphaeroides to obtain bacterial cells, mixing the bacterial cells with an organic solvent, stirring, standing for 40 to 60 minutes, centrifuging to obtain a supernatant organic phase, and vacuum drying the supernatant organic phase to obtain the oxidized coenzyme Q10.

8. The method according to claim 7, characterized in that The Rhodobacter sphaeroides is Rhodobacter sphaeroides INGIA-NM002 with a deposit number of CGMCC No. 32823.

9. The method according to claim 8, characterized in that The preparation method of the fermentation broth comprises: Step (1): inoculating the Rhodobacter sphaeroides INGIA-NM002 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, 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 the fermentation liquid.

10. The method according to claim 9, characterized in that The concentrations of the components of the solid culture medium are as follows: 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; 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 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; 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.

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