A method for de novo synthesis of reduced coenzyme Q10

Abstract

The present invention relates to the field of biotechnology, and particularly to a method for de novo synthesis of reduced coenzyme Q10. The method provided by the present invention is based on the preparation of coenzyme Q10 reductase from five sources using oxidized coenzyme Q10 as a substrate. The oxidized coenzyme Q10 can be de novo synthesized by Rhodobacter sphaeroides INGIA-NM002 under special condition parameters. The whole technological process has the advantages of high yield, high purity, low cost, high safety, and being more suitable for industrial production, etc.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a method for de novo synthesis of reduced coenzyme Q10. Background Art

[0002] Coenzyme Q10 (CoQ10) 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 is involved in energy production. CoQ10 has two forms: oxidized and reduced. Reduced coenzyme Q10 (Ubiquinol) has high bioavailability and good oral absorbability, and has functions such as antioxidant (scavenging free radicals, etc.), preventing vascular atherosclerosis, and improving chronic respiratory diseases. It has a wide range of applications in the fields of food (including beverages), nutritional products (including nutritional supplements), feeds, daily chemical products (including cosmetics), etc.

[0003] Traditional production methods of reduced coenzyme Q10 include synthesis, fermentation, extraction from natural products, etc. Currently, the commonly used process for producing reduced coenzyme Q10 is to reduce oxidized coenzyme Q10 using reducing agents 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 agents 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 has 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 restricted.

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

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

[0006] To achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0007] The present invention provides a method for de novo synthesis of reduced coenzyme Q10, including: 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 synthesis of oxidized coenzyme Q10 in the above method includes: synthesizing oxidized coenzyme Q10 using Rhodobacter sphaeroides capable of synthesizing oxidized coenzyme Q10.

[0009] In some specific embodiments of the present invention, the above method includes: using glucose as a substrate, synthesizing oxidized coenzyme Q10 using Rhodobacter sphaeroides capable of synthesizing oxidized coenzyme Q10, and then using the oxidized coenzyme Q10 as a substrate, synthesizing reduced coenzyme Q10 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.

[0010] In some specific embodiments of the present invention, the NCBI GenPept accession number of the coenzyme Q10 reductase 1 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; the UniProtKB accession number of the coenzyme Q10 reductase 5 is A0A067XMP1.1.

[0011] In some specific embodiments of the present invention, the above method includes: 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, and 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, cultivate the Rhodobacter sphaeroides to obtain a fermentation broth, break the fermentation broth, and then mix it with (a) Tris-HCl, (b) NaCl, (c) NADH or NADPH, and (d) reductase, and react 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;

[0013] The reductase is coenzyme Q10 reductase 1, coenzyme Q10 reductase 2, coenzyme Q10 reductase 3, coenzyme Q10 reductase 4, or 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%-75%, 35%-65%, 40%-60%, 45%-55%, or 50%;

[0016] The preparation method of the reductase includes: synthesizing an expression vector, the expression vector having the 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 to obtain a recombinant strain, culturing the recombinant strain, and when the OD 600 reaches 0.5-0.7 or 0.55-0.65, harvest the recombinant strain broth and collect the recombinant strain cells, wash the recombinant strain cells, resuspend, break the recombinant strain cells, and centrifuge to take the supernatant to obtain the reductase.

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

[0018] In some specific embodiments of the present invention, the Rhodobacter sphaeroides of the above method is Rhodobacter sphaeroides INGIA - NM002 with a preservation 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 includes:

[0020] Step (1): Inoculating the Rhodobacter sphaeroides INGIA - NM002 into a solid medium, culturing to obtain single colonies;

[0021] Step (2): Inoculating the single colonies into a seed medium, culturing to obtain a seed solution;

[0022] Step (3): Inoculating the seed solution into a fermentation medium, the fermentation medium contains VB1 and ferrous sulfate. When inoculating, the concentration of VB1 can be 10 - 30 ppm or 15 - 25 ppm, the concentration of ferrous sulfate can be 1 - 3 g / L or 0.5 - 2.5 g / L, and the fermentation time can be 58 - 62 h or 59 - 61 h;

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

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

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

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

[0027] In some specific embodiments of the present invention, the tank pressure during the fermentation described 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 during the fermentation described 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 during the fermentation described 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 Rhodobacter sphaeroides INGIA - NM002.

[0031] In some specific embodiments of the present invention, the preparation concentrations of the components of the above solid medium 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 medium includes 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 dry corn steep liquor; 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 niacin; 0.7 - 1.3 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 - 1.3 g / L or 0.9 - 1.1 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 dry 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 - 1.1 ppm VB2; 0.7 - 1.3 ppm or 0.9 - 1.1 ppm nicotinic acid; 0.4 - 0.6 ppm or 0.45 - 0.55 ppm biotin; 0.7 - 1.3 ppm or 0.9 - 1.1 ppm folic acid.

[0034] In some specific embodiments of the present invention, during the fermentation in step (3) of the above method, a feeding step is further included;

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

[0036] The feed for primary feeding comprises 500 - 700 g / L or 550 - 650 g / L glucose, 15 - 25 g / L or 18 - 22 g / L magnesium sulfate heptahydrate;

[0037] The feed for secondary feeding comprises 125 - 375 g / L potassium dihydrogen phosphate.

[0038] In some specific embodiments of the present invention, during the fermentation in steps (3) and (4) of the above method, concentrated ammonia water is used to adjust the pH to between 6.8 - 7.0 or 6.85 - 6.95.

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

[0040] Experiments show that when the coenzyme is NADH, the conversion rates of reduced coenzyme Q10 of coenzyme Q10 reductase expressed by 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. When the coenzyme is NADPH, the conversion rates of reduced coenzyme Q10 of coenzyme Q10 reductase expressed by 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 conversion rate of reduced coenzyme Q10 of 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 to combine with the fermentation products of ecCoQH-1, ecCoQH-2, ecCoQH-3, ecCoQH-4, or ecCoQH-5 to achieve the de novo synthesis of reduced coenzyme Q10, which has high yield, high purity, low cost, and high safety, and is more suitable for industrial production. Brief Description of the Drawings

[0041] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.

[0042] Figure 1 Showing the protein electrophoresis results of coenzyme Q10 reductase 1 derived from Deltaproteobacteria bacterium;

[0043] Figure 2 Showing the protein electrophoresis results of coenzyme Q10 reductase 3 derived from Pseudocercospora eumusae;

[0044] Figure 3 Showing the protein electrophoresis results of the reductase. Among them, 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 4Show the conversion rate of reduced coenzyme Q10 of different strains when the coenzyme is NADH;

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

[0047] Figure 6 Show the conversion rate of reduced coenzyme Q10 of ecCoQH-1 at different temperatures.

[0048] Biological deposit description

[0049] Biological material: INGIA-NM002, taxonomic name: Rhodobacter sphaeroides, deposited with the General Microbiological 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

[0050] The present invention discloses a method for de novo synthesis of reduced coenzyme Q10. Those skilled in the art can draw on 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. 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.

[0051] This application provides a method for producing reduced coenzyme Q10. This method uses the following coenzyme Q10 reductase as a reducing agent to reduce oxidized coenzyme Q10 to produce reduced coenzyme Q10. The coenzyme Q10 reductase includes any one of the following.

[0052] Coenzyme Q10 reductase 1 derived from Deltaproteobacteria bacterium, NCBI GenPept accession number is MBP78919.1, and the nucleotide sequence optimized for E. coli codons is:

[0053]

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

[0055]

[0056] Coenzyme Q10 reductase 3 derived from Pseudocercospora eumusae, NCBI GenPept accession number: KXS94104.1, and the nucleotide sequence optimized for Escherichia coli codons is as follows:

[0057]

[0058] Coenzyme Q10 reductase 4 derived from Nannizzia gypsea CBS 118893, with NCBI GenPept accession number XP_003169128.1, and the nucleotide sequence optimized for Escherichia coli codons is as follows:

[0059]

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

[0061]

[0062] As a control for this application, this application also relates to a reductase derived from Caldalkalibacillus thermarum, with NCBI GenPept accession number WP_007502350.1 and nucleotide sequence as follows:

[0063]

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

[0065]

[0066] In this application, both 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 ordinary commercially available products and can be purchased from the market.

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

[0069] Example 1: Strain construction

[0070] 1. Construction of recombinant plasmid

[0071] Construct a 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 synthesized in full. 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] Taking the construction of bacteria ecCoQH-1 expressing reductase as an example:

[0080] Mix the above pHQ-1 plasmid (100 ng) with 100 μL of BL21(DE3) competent cells. After electrotransformation, transfer them to LB medium and culture for 30 min. Then evenly spread them on an LB plate containing the antibiotic kanamycin (50 mg / L) and culture overnight. Pick single colonies for preservation to obtain the bacterium ecCoQH-1 expressing reductase.

[0081] The construction methods of the other bacteria expressing reductase are the same as that of ecCoQH-1, and the bacteria ecCoQH-2, ecCoQH-3, ecCoQH-4, and ecCoQH-5 expressing reductase are obtained respectively.

[0082] 3. Construction of control bacteria

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

[0084] After mixing the above pHQ-ctrl plasmid (100 ng) with 100 μL of BL21(DE3) competent cells, transfer them to LB medium by electrotransformation and culture for 30 min. Then evenly spread them on an LB plate containing the antibiotic kanamycin (50 mg / L) and culture overnight. Pick single colonies for preservation to obtain the bacterium ecCoQH-c expressing reductase.

[0085] Example 2: Verification and comparison of enzyme activity

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

[0087] Streak the constructed strains ecCoQH-1, ecCoQH-2, ecCoQH-3, ecCoQH-4, and ecCoQH-5 on an LB plate without resistance. After culturing overnight at 37 °C, pick single colonies into 5 mL of LB medium and culture at 37 °C and 200 rpm until OD 600 ~0.6. Then add 0.1 mM of IPTG for induction and culture in a shaker at 22 °C for 18 h, and then harvest the bacteria. The obtained bacterial solution is washed 3 times with physiological saline, resuspended in 1 mL of physiological saline, and then put into an ultrasonic crusher for sufficient disruption. After centrifugation (12,000 rpm, 1 min), take the supernatant 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 patterns are shown in Figure 1 , Figure 2 , Figure 3 .

[0088] The constructed ecCoQH-c strains of the control group were streaked on LB plates without resistance. After overnight culture at 37°C, single colonies were picked and inoculated into 5 mL of LB medium, and cultured at 37°C and 200 rpm until the OD 600 ~0.6. Then, 0.1 mM IPTG was added for induction, and the culture was continued in a shaker at 22°C for 18 h before harvesting the bacteria. The obtained bacterial solution was washed 3 times with normal saline and resuspended in 1 mL of normal saline. Then, it was placed in an ultrasonic crusher for sufficient disruption. After centrifugation (12,000 rpm, 1 min), the supernatant was taken 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, and NADH was added as the 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, that is, in a glove box filled with nitrogen. All sample solutions were degassed under nitrogen before use. The reaction temperature was set at 37°C.

[0092] 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 Figure 4 shown. The specific data are shown in Table 1.

[0093] Table 1: Conversion rates 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, and NADPH was added as the 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, that is, in a glove box filled with nitrogen. All sample solutions were degassed under nitrogen before use. The reaction temperature was set at 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 Figure 5 shown. 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] The HPLC detection conditions are as follows: chromatographic column: C18; mobile phase: methanol:ethanol = 1:1, flow rate: 1 mL / min, wavelength: 275 nm; sample injection: 20 μL; detection temperature: 30 °C.

[0100] From Figure 4 and 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] Select the ecCoQH-1 with the highest conversion rate above as the strain for the temperature optimization experiment, and its protein expression step and reaction step are as described above.

[0103] The reaction temperatures are set at 22 °C, 30 °C, 37 °C, 45 °C, 50 °C, 60 °C, and 70 °C respectively. The conversion rate results are as Figure 6 shown, and 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 and Table 3, it can be seen that the conversion rate is the highest when the reaction temperature is 45 °C.

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

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

[0109] 1. Production of oxidized coenzyme Q10

[0110] S101. 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;

[0111] S102. Seed culture: Pick the single colonies after activation in step S101 and inoculate them into the seed medium. Specifically, each 500 mL shake flask is filled with 100 mL of liquid, and 3 - 5 single colonies are picked and cultured at 200 rpm and 30 - 35 °C for 24 - 30 h to obtain the seed liquid;

[0112] S103. Fermentation culture: Inoculate the seed liquid obtained in the above step S102 into the fermentation basal medium, with an inoculation amount of 10%. The initial culture conditions are 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, a ferrous sulfate concentration of 2 g / L, and a VB1 concentration of 20 ppm; After about 4 - 5 h of fermentation, feed supplement 2 is added. After 6 - 8 h of fermentation, the sugar is exhausted and the pH rises, and then feed supplement 1 is added. Samples are taken every 4 h to measure sugar and phosphorus. The sugar concentration is 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 in the later stage is controlled at 5 g / L, and the residual phosphorus concentration in the later stage is 20 ppm. The aeration ratio is 0.2 and the dissolved oxygen is 10%. The pH value of the fermentation broth is adjusted between 6.7 - 7.1 by adding ammonia water throughout the process. When the staining of the bacteria becomes lighter, some hyphae autolyze, and the yield growth slows down, the fermentation is terminated. Among them, the "early stage" and "later stage" are distinguished with 60 h as the boundary.

[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, B: anhydrous ethanol, ratio: 65:35.

[0114] The formula of the above - used medium is as follows:

[0115] 1) Solid medium

[0116] Table 4: Components of the solid medium

[0117]

[0118] 2) Seed medium

[0119] Table 5: Components of the seed medium

[0120]

[0121] 3) Fermentation medium

[0122] Table 6: Components of the fermentation basal medium

[0123]

[0124] Table 7: Supplementary liquid (filtered and sterilized separately and added after calculating based on the initial fermentation volume)

[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, used to control pH.

[0132] 2. Production of reduced coenzyme Q10

[0133] Method 1 (using the purified oxidized coenzyme Q10 from the fermentation broth of the deposited strain as the substrate):

[0134] S201. After the fermentation is completed, take 10 mL of the fermentation broth after the completion of step S103 above, centrifuge to obtain the cell precipitate, slowly add 10 - 20 mL of an organic solvent (such as petroleum ether or ethyl acetate, petroleum ether is used in this example) to the cell precipitate, stir to dissolve thoroughly, let stand for 40 - 60 min, then centrifuge at 12,000 rpm to obtain the supernatant organic phase, vacuum rotary evaporate the organic phase at 0.1 - 0.5 MPa to obtain a powder, which is the target precursor substance (oxidized coenzyme Q10), and add 10 mL of petroleum ether to dissolve it;

[0135] S202. Take the coenzyme Q10 reductase 1 and the reductase of the control bacteria prepared above respectively and the target precursor substance (oxidized coenzyme Q10) in step S201, add NADH as a coenzyme, mix thoroughly, the final volume of the reaction mixture is 2 mL, containing: 100 mM Tris-HCl (pH = 7.6), 500 mM NaCl, 2 mM NADH, 1 mL of reductase and 1 mM of the 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, and all sample solutions are degassed under nitrogen before use. The reaction temperature is set at 45°C. After the reaction proceeds for 30 min, quench with methanol, and then quickly perform liquid chromatography to determine the content and calculate the conversion rate.

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

[0137] S301. Take 10 mL of the fermentation broth after the fermentation in the above step S103 and centrifuge to obtain a precipitate. Resuspend it with 2 mL of normal saline, then put it into an ultrasonic crusher and perform sufficient crushing (stop for one second every 4 seconds, for a total of 30 minutes) to obtain a precursor fermentation broth containing the target precursor substance (oxidized coenzyme Q10).

[0138] S302. Add the coenzyme Q10 reductase 1 and the control bacterium reductase prepared above to the precursor fermentation broth containing the target precursor substance (oxidized coenzyme Q10) in step S301 respectively. Add NADH as a coenzyme and 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 of reductase and the precursor fermentation broth (containing 1 mM oxidized coenzyme Q10, and the addition amount is calculated based on the HPLC quantification result: take 500 μL of the precursor fermentation broth and mix it with an equal volume of a mixture of petroleum ether and ethyl acetate, where the volume ratio of petroleum ether to ethyl acetate is 4:1. After extraction, send it for HPLC quantification to obtain the concentration of oxidized coenzyme Q10 in the precursor fermentation broth, and determine the addition amount according to the concentration). 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. The reaction temperature is set at 45 °C. After the reaction proceeds for 30 min, quench it with methanol, and then quickly perform liquid chromatography to determine the content and calculate the conversion rate.

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

[0140] Table 10: Conversion rates of reduced coenzyme Q10 of Method 1 and Method 2

[0141]

[0142] It can be seen that the oxidized coenzyme Q10 obtained by purifying the fermentation broth fermented by the preserved strain INGIA-NM002 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 conversion rate of the reductase of the control bacterium; and it can be found that directly using the crushed fermentation broth as a substrate and synthesizing reduced coenzyme Q10 with the coenzyme Q10 reductase 1 of the present invention can reach a conversion rate of 54.5%, while the conversion rate of the reductase of the control bacterium 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 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 still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for synthesizing reduced coenzyme Q10 from scratch, characterized in that: include: synthesizing oxidized coenzyme Q10, and then using the oxidized coenzyme Q10 as a substrate to synthesize reduced coenzyme Q10 based on coenzyme Q10 reductase; The coenzyme Q10 reductase is derived from Deltaproteobacteria bacteria , the encoding gene is SEQ ID NO:

1.

2. The method according to claim 1, characterized in that The synthesis of oxidized coenzyme Q10 comprises: using a spherical red bacterium ( Rhodobacter sphaeroides ) synthesizes oxidized coenzyme Q10.

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

4. The method according to claim 3, 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.

5. The method according to claim 4, characterized in that The volume ratio of the coenzyme Q10 reductase to the reaction system is 25% to 75%; The preparation method of the coenzyme Q10 reductase comprises: synthesizing an expression vector, wherein the expression vector has a sequence shown in SEQ ID NO: 1, introducing the expression vector into competent cells and culturing to obtain a recombinant strain, culturing the recombinant strain, and waiting for the OD value of the recombinant strain to reach 1. 600 When the concentration reaches 0.5-0.7, the recombinant strain bacterial liquid is harvested and the recombinant strain bacterial cells are collected, the recombinant strain bacterial cells are washed, resuspended, and the recombinant strain bacterial cells are broken, and the supernatant is taken by centrifugation to obtain the coenzyme Q10 reductase.

6. The method according to claim 2, characterized in that The preparation method of oxidized coenzyme Q10 comprises: Rhodobacter sphaeroides ) is centrifuged to obtain bacterial cells, the bacterial cells are mixed with an organic solvent, stirred, and allowed to stand for 40 to 60 min, and the supernatant organic phase is centrifuged to obtain a vacuum spin dryer to obtain oxidized coenzyme Q10.

7. The method according to claim 6, characterized in that The spherical red bacteria ( Rhodobacter sphaeroides ) is a spherical red bacterium with a deposit number of CGMCC No. 32823 ( Rhodobacter sphaeroides )INGIA-NM002.

8. The method according to claim 7, characterized in that The method for preparing the fermentation broth comprises: Step (1): the Rhodobacterium sphaeroides ( 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 the fermentation liquid.

9. The method according to claim 8, 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.

Images