CN113736680B

CN113736680B - Autotrophic Pseudonocardia and its application

Info

Publication number: CN113736680B
Application number: CN202010480265.1A
Authority: CN
Inventors: 杨虎; 丰亚辉; 王仲清; 张克闯; 林伟; 罗忠华; 黄芳芳
Original assignee: Dongguan Dongyangguang Generic Research And Development Co ltd; Guangdong HEC Pharmaceutical
Current assignee: Dongguan Dongyangguang Generic Research And Development Co ltd; Guangdong HEC Pharmaceutical
Priority date: 2020-05-30
Filing date: 2020-05-30
Publication date: 2024-11-26
Anticipated expiration: 2040-05-30
Also published as: CN113736680A

Abstract

The invention relates to autotrophic Pseudonocardia and application thereof, and belongs to the technical field of microorganisms. The autotrophic Pseudonocardia can be used for preparing calcitriol and/or calcitriol, and the yield is remarkably improved compared with the original strain.

Description

Autotrophic Pseudonoco Carlsberg and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to autotrophic Pseudonocardia and application thereof.

Background

The Pseudonocardiaceae is an actinomycete which has similar evolution and different forms, contains Meso-DAP (Meso-diaminopimelic acid) in peptidoglycan, contains galactose and arabinose in whole cell hydrolysate and does not contain mycolic acid. Many members of this family are capable of fermentation to produce important biologically active substances, including antibiotics, enzymes, vitamins, and the like. Meanwhile, the family has few pathogenic bacteria and is a safe microorganism.

Calcitonin, also known as 25-hydroxyvitamin D3, has been loaded into the united states pharmacopeia USP36-NF31 and European Pharmacopeia (EP) 8.0 as one of the representatives of the active vitamin D3 class of drugs; the composition is mainly used for treating bone disorder, metabolic bone diseases and the like clinically, and is especially suitable for patients with normal renal function and liver dysfunction. Calcitriol (Calcitriol), also known as 1, 25-dihydroxyvitamin D3, can promote calcium absorption in intestinal tract, and increase blood calcium level, and can be used for treating parathyroid hypofunction, vitamin D dependent rickets, etc. Research shows that the active vitamin D3 medicine and its analogues have the pharmacological activity of regulating target cell, inducing cell differentiation and inhibiting cell proliferation, and may be used in treating malignant tumor, psoriasis, immunological diseases, etc.

The preparation of the calcite glycol comprises two methods of chemical synthesis and biological fermentation, wherein the chemical synthesis reaction has a plurality of steps, expensive reagents are needed, racemes can be generated in the reaction process, resolution is needed, the process is complex, the yield is greatly influenced, the completion of resolution is difficult, and the environment is greatly polluted by the production of the calcite glycol by chemical synthesis. The microbial fermentation mode can utilize the enzyme specificity in microbial cells to solve the chiral structure problem of the calcitonin, and the method has the advantages of mild production conditions, high yield, low cost and environmental friendliness, and is an effective alternative chemical synthesis method. However, the concentration of the substrate vitamin D3 added in the prior art is low, and the maximum concentration of vitamin D3 added in the method disclosed in patent US5474923 is only 1000ug/mL, which is disadvantageous in that the yield of calcitol is increased by increasing the dosage of vitamin D3.

Therefore, a method for preparing calcitol by using a biological fermentation method is still needed to be researched, so that the method is simple and convenient to operate, easy to implement, high in yield, high in purity, low in cost and environment-friendly.

Disclosure of Invention

The invention provides autotrophic Pseudonocardia and application thereof, in particular to autotrophic Pseudonocardia G-5H1 and a method for preparing calcitriol and/or calcitriol by using the same.

The invention carries out strain evolution based on ultraviolet mutagenesis and ribosome engineering mutagenesis on an original strain with a preservation number of CGMCC 4.1211 and a name of Pseudonocardia autotrophica of the autotrophic Pseudonocardia, screens and obtains a new high-yield autotrophic Pseudonocardia G-5H1 which is preserved in China general microbiological culture Collection center of Beijing at a date of 09 in 2020 and has a preservation number of CGMCC No.19461 and is classified and named as: pseudonocardia autotrophica G-5H1. The invention also establishes a two-phase fermentation method for preparing calcitriol and/or calcitriol by using the obtained autotrophic Pseudonocardia G-5H1, and the yield is obviously improved.

In one aspect, the invention provides an autotrophic Pseudonocardia G-5H1, which has been deposited in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, having a collection number of CGMCC No.19461, and a classification designation of Pseudonocardia autotrophica G-5H1, wherein the collection number is China general microbiological culture Collection center of Beijing, and the 09 th year of the year 2020.

The autotrophic Pseudonocardia G-5H1 is obtained by performing strain evolution based on ultraviolet mutagenesis and ribosome engineering mutagenesis on an original strain of the autotrophic Pseudonocardia with a preservation number of CGMCC 4.1211 and a name of Pseudonocardia autotrophica, and screening.

For ultraviolet mutagenesis and/or ribosome engineering mutagenesis, the original strain of Pseudonocardia autotrophica (CGMCC 4.1211, titled: pseudonocardia autotrophica) is cultured to obtain spore suspension, and the spore suspension with known spore concentration is obtained by dilution and counting, for example, glycerin aqueous solution is used as solvent to obtain spore suspension with the concentration of 10 ⁶ spores/ml. The spore suspension of known spore concentration is subjected to uv mutagenesis and/or ribosome mutagenesis under a variety of conditions, depending on the result, corresponding relatively favourable mutagenesis conditions are obtained. By using the obtained relatively favorable mutagenesis conditions, ultraviolet mutagenesis and ribosome engineering mutagenesis can be performed to obtain mutant strains. After obtaining the mutant strain, a preferred high-yielding strain can be obtained by screening.

In another aspect, the present invention also provides a method for screening high-yielding strains including autotrophic nocardia G-5H1, which can be screened at high throughput to obtain preferred high-yielding strains. The G-5H1 strain can be obtained by a screening method through a mutant strain obtained by ultraviolet mutagenesis and/or ribosome engineering mutagenesis of the autotrophic Nocardia (collection number CGMCC 4.1211, name: pseudonocardia autotrophica).

A method of screening for high yielding strains including autotrophic nocardia G-5H1 comprising:

(1) Inoculating single colonies of mutant strains obtained by ultraviolet mutagenesis and/or ribosome engineering mutagenesis of original strains into an orifice plate with a Gaoshi No. 1 culture medium, culturing at a constant temperature of 25-30 ℃ for at least 7 days, transferring to a fermentation culture medium orifice plate, shaking and culturing the microplates for 3-7 days, preferably 5 days, centrifugally collecting thalli, washing with PBS to remove the culture medium, adding veratryl alcohol and PBS, reacting for 0.1-2 h at 25-30 ℃, adding 2,4 dinitrophenylhydrazine, and reacting for 10-16 h at 25-30 ℃; then, according to the color change, selecting colonies with colors changed from yellow to orange or darkened, and obtaining potential high-yield strains;

(2) Shake flask fermentation of the resulting potential high yielding strain: inoculating single colonies of the potential high-yield strain into a seed culture medium respectively, and carrying out shaking culture at a constant temperature of 25-30 ℃ for 48-72 hours, preferably 60 hours; then, according to the volume ratio of the seed culture medium to the fermentation culture medium and the inoculum size of 5-10 percent of the volume ratio, transferring the mixture into the fermentation culture medium (such as the original fermentation culture medium or the optimized fermentation culture medium), and culturing the mixture in a shaking table at the constant temperature of 25-30 ℃ for 3-7 days, preferably 4 days; then adding a substrate vitamin D3 into the strain, reacting for 1-5 days, preferably 3 days, detecting and analyzing the product by using HPLC, and screening out corresponding strains with high product yield; obtaining the high-yield strain.

In some embodiments, a method of screening for high-yielding strains including autotrophic nocardia G-5H1 comprises:

(1) Inoculating a single strain of a mutant strain obtained by ultraviolet mutagenesis and/or ribosome engineering mutagenesis of an original strain into a 96-well plate with 300 mu L of culture medium/well of Gaoshi No. 1 culture medium, culturing at a constant temperature of 25-30 ℃ for at least 7 days, transferring the 96-well plate liquid fermentation culture medium, adding 180-270 mu L of fermentation culture medium into each well, shaking and culturing a micro-plate at 400-600 rpm for 3-7 days, preferably 5 days, centrifuging the micro-plate by using a micro-plate centrifuge to collect thalli, washing the culture medium by using PBS, adding 50-70 mu L of 8-12 mmol/L veratrole and 100 mu L-140 mu L of PBS into each well, reacting for 0.5-1.5 hours at 25-30 ℃, adding 0.05-0.1 mu L of 2,4 dinitrophenylhydrazine, and reacting for 10-16 hours at 25-30 ℃; then, according to the color change, selecting colonies with colors changed from yellow to orange or darkened, and obtaining potential high-yield strains;

(2) Shake flask fermentation of the resulting potential high yielding strain: inoculating single colonies of the potential high-yield strain into a seed culture medium respectively, and carrying out shaking culture at constant temperature of 25-30 ℃ and 220-280 rpm for 48-72 hours, preferably 60 hours; then, the seed culture medium and the fermentation culture medium are transferred into the fermentation culture medium (such as the original fermentation culture medium or the optimized fermentation culture medium) according to the volume ratio of the seed culture medium to the fermentation culture medium, and the seed culture medium and the fermentation culture medium are subjected to shaking culture at a constant temperature of between 25 and 30 ℃ and between 220 and 280rpm for 3 to 7 days, preferably 4 days; then adding a substrate vitamin D3 into the strain, reacting for 1-5 days, preferably 3 days, detecting and analyzing the product by using HPLC, and screening out corresponding strains with high product yield; obtaining the high-yield strain.

(1) Inoculating a single strain of a mutant strain obtained by ultraviolet mutagenesis and/or ribosome engineering mutagenesis of an original strain into a 96-well plate with 300 mu L of culture medium/well of Gaoshi No. 1 culture medium, culturing at a constant temperature of 25-30 ℃ for at least 7 days, transferring the 96-well plate liquid fermentation culture medium, adding 240 mu L of fermentation culture medium into each well, shaking and culturing by a micro-plate shaker at 500rpm for 3-7 days, preferably 5 days, centrifuging the culture medium by a micro-plate centrifuge, washing the culture medium by using PBS, adding 60 mu L of 10mmol/L veratryl alcohol and 120 mu L of PBS into each well, reacting for 1h at 25-30 ℃, and then adding 1 mu L of 0.08g/L of 2,4 dinitrophenylhydrazine, and reacting for 10 hours-16 hours at 25-30 ℃; then, according to the color change, selecting colonies with colors changed from yellow to orange or darkened, and obtaining potential high-yield strains;

(2) Shake flask fermentation of the resulting potential high yielding strain: single colonies of potential high-yield strains are respectively inoculated into a seed culture medium, and the culture is carried out for 48 to 72 hours, preferably 60 hours at a constant temperature of 25 to 30 ℃ and a constant speed of 250 rpm; then, the seed culture medium and the fermentation culture medium are transferred into the fermentation culture medium (such as the original fermentation culture medium or the optimized fermentation culture medium) according to the volume ratio of the seed culture medium to the fermentation culture medium, and the seed culture medium and the fermentation culture medium are subjected to shaking culture at the constant temperature of 25-30 ℃ and at the constant speed of 250rpm for 3-7 days, preferably 4 days; then adding a substrate vitamin D3 into the strain, reacting for 1-5 days, preferably 3 days, detecting and analyzing the product by using HPLC, and screening out corresponding strains with high product yield; obtaining the high-yield strain.

The screening method can rapidly screen potential high-yield strains or high-yield strains. In the above screening method, the fermentation medium may be an original fermentation medium or an optimized and modified fermentation medium. The concentrations of the components or formulation components of the original fermentation medium are: 15g/L soybean peptone, 15g/L glucose, 5g/L yeast powder, 10g/L fish meal, 5g/L NaCl (sodium chloride), 0.1g/L K ₂HPO₄ (dipotassium hydrogen phosphate), 0.1g/L NaF (sodium fluoride) and 2g/L CaCO ₃ (calcium carbonate) calculated according to the total volume of the fermentation medium; the pH value is controlled to 7.0, and the balance is water.

The inventors have obtained a plurality of high-yielding strains including the G-5H1 strain by screening, wherein the yield of the G-5H1 strain is relatively highest. Gene sequencing of the obtained high-yield strain found that: the rpsL gene of the G-5H1 strain (30S ribosomal protein S12 ) is mutated at a number of positions, including its 9-nucleotide, 88-amino acid and other positions, as compared to the rpsL of the original strain. The strain G-5H1 is preserved in China general microbiological culture collection center of Beijing (CGMCC) in the 09 th year 2020, and has a preservation number of CGMCC No.19461 and a name of Pseudonocardia autotrophica G-5H1.

In another aspect, the invention provides an application of autotrophic Pseudonocardia G-5H1 in preparing calcitriol and/or calcitriol.

In some embodiments, the use is the preparation of calcitol by a two-phase fermentation process.

In some embodiments, the use is the preparation of calcitriol by a two-phase fermentation process.

Different fermentation media have different fermentation effects and different effects on the generation of target products.

On the other hand, the inventors found that the above-mentioned primary fermentation medium is used in the above-mentioned two-phase fermentation process for preparing calcitriol or calcifediol, and the yield of the target product is relatively low, and thus the primary fermentation medium can be optimally improved. The inventor optimizes the main components of the original fermentation medium, such as carbon source, nitrogen source, inorganic salt and the like, and discovers that the optimized fermentation medium can improve the yield of target products.

For optimal improvement of the carbon source, the inventors used glucose, fructose, mannitol, glycerol, maltose, dextrin, corn flour, corn steep liquor, soluble starch, galactose, lactose, sorbitol, xylose and sucrose as initial carbon sources, respectively, and evaluated the conversion of calcitol under each condition. The results show that the conversion rate can be better improved when the sucrose and the glycerol are used as single carbon sources, and the sucrose is used as the first quick-acting carbon source of the fermentation medium in consideration of the fact that the price of the glycerol is about 2 times that of the sucrose. And determining the corn steep liquor as a second long-acting carbon source through multiple optimization. In view of the seed engagement, it is advantageous to add 5g/L glucose. After a series of test comparisons, the combination of 10g/L sucrose, 5g/L glucose and 15.37g/L corn steep liquor was determined to be the preferred combination.

After determining the preferred carbon source composition, the inventors compared 14 common nitrogen sources including peptone, tryptone, yeast powder, soy peptone, malt extract, beef extract, enzymatic casein, corn steep liquor, corn flour, soybean meal, cotton seed meal, soluble fish meal, L-tyrosine and phytone peptone (Phytone, BD company) for the purpose of optimizing the nitrogen source. The results show that tryptone, yeast powder, soybean peptone and soybean cake powder can well improve the conversion rate; the multi-element combination optimization shows that the combination of yeast powder and soybean peptone has obvious advantages in the aspects of potency, production cost and the like. After a series of test comparisons, the combination of 35g/L soytone and 31.5g/L yeast powder was determined to be the preferred combination.

Further, the inventors conducted comparative tests on NaCl, KCl (potassium chloride), mgSO ₄ (magnesium sulfate), feSO ₄ (ferrous sulfate), caCl ₂ (calcium chloride), mnCl ₂ (manganese dichloride), (NH ₄)₂SO₄ (ammonium sulfate), K ₂HPO₄, naF, etc., and as a result, the addition of MgSO ₄ and FeSO ₄ could significantly improve the conversion rate of calcitol (this is not excluded because the enzymes involved in the conversion need Mg ² ⁺ and Fe ²⁺), phosphate could promote the growth of cells, naCl plays an important role in the electron transfer of respiratory chain, and a certain amount of CaCO ₃ could balance the pH value of the fermentation broth and facilitate the growth of mycelium.

Further, in view of the problem of extremely low solubility of the substrate and the product, the inventors have selected a cosolvent, a surfactant, an agent promoting formation of a membrane channel, etc. which may be added to the medium, including tween 20, tween 80, triton X-100, mannitol, sorbitol, betaine, PEG4000, nisin, sodium dodecyl sulfate, caCl ₂, beta-cyclodextrin, hydroxypropyl-cyclodextrin, and sulfobutyl-cyclodextrin. The test result shows that the effects of the surfactants Tween 20, tween 80 and sorbitol are obvious, and the cosolvent beta-cyclodextrin is good. The result of the multi-element combination optimization experiment shows that the combination of 5mL/L Tween 20 and 5g/L beta-cyclodextrin is obviously superior to other combinations.

Thus, the present invention provides an optimized fermentation medium that is more conducive to fermentation and production of the desired product.

An optimized fermentation medium comprising or formulated with components comprising: sucrose, glucose, corn steep liquor, soybean peptone, yeast powder, feSO ₄,MgSO₄,CaCO₃,NaCl,K₂HPO₄, naF, tween 20 and water; the pH value is 6.5-7.5, preferably 7.0; optionally including beta-cyclodextrin; is beneficial to the fermentation and the preparation of calcitriol and/or calcitriol of the autotrophic Pseudonocardia G-5H 1.

In some embodiments, an optimized fermentation medium comprises the following components or formulations in the following concentrations of the components, calculated from the total volume of the fermentation medium: 8g/L-12g/L of sucrose, 3g/L-6g/L of glucose, 12g/L-18g/L of corn steep liquor, 30g/L-40g/L of soybean peptone, 25g/L-35g/L,FeSO₄ 0.05g/L-0.2g/L,MgSO₄ 0.1g/L-1g/L,CaCO₃ 1g/L-5g/L,NaCl 1g/L-10g/L,K₂HPO₄ 0.01g/L-0.5g/L,NaF 0.01g/L-0.5g/L and 201 ml/L-10ml/L of tween, wherein the pH value is 6.5-7.5, preferably 7.0; optionally including beta-cyclodextrin, which may be present in a concentration of 3g/L to 8g/L or 0g/L, the balance being water; is beneficial to the fermentation and the preparation of calcitriol and/or calcitriol of the autotrophic Pseudonocardia G-5H 1.

In some embodiments, according to some embodiments of the present invention, an optimized fermentation medium may be controlled to have a composition or formulation with a composition concentration of 10g/L sucrose, 5g/L glucose, 15.37g/L corn steep liquor, 35g/L soytone, 31.5g/L,FeSO₄ 0.1g/L,MgSO₄ 0.5g/L,CaCO₃ 2g/L,NaCl 5g/L,K₂HPO₄ 0.1g/L,NaF 0.1g/L ml yeast powder and 20 ml/L tween, a beta-cyclodextrin concentration of 0g/L or 5g/L, the balance water, a pH of 6.5 to 7.5, preferably 7.0, and the balance water, based on the total volume of the fermentation medium. The optimized fermentation medium is more beneficial to the fermentation and preparation of calcitriol and/or calcitriol by the autotrophic nocardia G-5H 1.

In some embodiments, according to some embodiments of the present invention, an optimized fermentation medium may be controlled to have a concentration of 10g/L sucrose, 5g/L glucose, 15.37g/L corn steep liquor, 35g/L soybean peptone, 31.5g/L,FeSO₄ 0.1g/L,MgSO₄ 0.5g/L,CaCO₃2g/L,NaCl 5g/L,K₂HPO₄ 0.1g/L,NaF0.1g/L g/L yeast powder, 5ml/L tween 20, pH 7.0, beta-cyclodextrin concentration of 0g/L or 5g/L, and the balance water, based on the total volume of the fermentation medium. This preferred fermentation medium is more advantageous for the fermentation and preparation of calcitriol and/or calcitriol by the autotrophic Pseudonocardia G-5H 1.

In some embodiments, an optimized fermentation medium is provided having a composition or formulation comprising 10g/L sucrose, 5g/L glucose, 15.37g/L corn steep liquor, 35g/L soytone, 31.5g/L,FeSO₄ 0.1g/L,MgSO₄ 0.5g/L,CaCO₃ 2g/L,NaCl 5g/L,K₂HPO₄ 0.1g/L,NaF0.1g/L g/L yeast powder and 5ml/L Tween 20, a pH of 7.0, and the balance water, based on the total volume of the fermentation medium. This preferred fermentation medium is more advantageous for the fermentation and preparation of calcitriol and/or calcitriol by the autotrophic Pseudonocardia G-5H 1.

The substrate vitamin D3 and the products calcitriol and calcitriol are fat-soluble and inhibit the growth of microorganisms, so that the bacteria, the substrate and the products are respectively placed in different phases, contact is reduced as much as possible, toxicity to the bacteria can be reduced, and the reaction and the generation and the acquisition of target products are facilitated. Therefore, a two-phase reaction system can reduce the influence of poor water solubility of a substrate, reduce toxicity of the substrate and a product to thalli, promote the utilization rate of the thalli and promote the reaction, and is favorable for the generation of the product.

In some embodiments, to obtain and determine the reaction system most favorable for the formation of the target product, the inventor compares and examines the reaction system formed by n-hexane, n-heptane, cyclohexane, ethanol, ethyl acetate, isopropanol, soybean oil, peanut oil or corn oil and the fermentation medium, and experimental results show that the two-phase reaction system of the n-heptane and the fermentation medium is more favorable for the formation of the target product.

The volume ratio of n-heptane to fermentation medium may be 1:9 to 1:2. In some embodiments, the volume ratio of n-heptane to fermentation medium may be 1:8-1:3 or 1:8-1:4. In some embodiments, the conversion is significantly increased at a volume ratio of n-heptane to fermentation medium of 1:5.

After determining the reaction system, the inventors have also examined the fermentation time and the reaction time. The inventor finds that the original yield of the target product can be relatively high by adopting the same or similar process and using the original strain and the original fermentation medium for 7 days and 5 days of fermentation; the strain G-5H1 and the original fermentation medium are used for fermenting for 5 days and reacting for 4 days, so that the yield of the obtained target product is obviously improved; the strain G-5H1 and the optimized and improved fermentation medium are used for fermenting for 5 days and reacting for 4 days, so that the yield of the obtained target product is obviously and remarkably improved. Finally, the fermentation is confirmed to be carried out for 4-7 days, preferably 5 days, and then the reaction is carried out for 3-5 days, preferably 4 days, so that the method is a relatively good fermentation reaction mode and is more beneficial to the generation of target products.

In another aspect, the invention also provides a method for preparing calcitriol and/or calcitriol, which comprises the step of preparing calcitriol and/or calcitriol by using autotrophic Nocardia G-5H1 and adopting a two-phase fermentation method.

The two-phase fermentation process comprises: (1) Inoculating the autotrophic Pseudonocardia G-5H1 into a seed culture medium, and culturing in a shaking table; then transferring and inoculating in fermentation medium (original fermentation medium or optimized fermentation medium), shake culturing; (2) Adding vitamin D3 and n-heptane, optionally adding beta-cyclodextrin, and reacting; and (5) after the reaction is finished, carrying out post-treatment to obtain a target product.

In the two-phase fermentation process, the fermentation medium may be the original fermentation medium described above or an optimized fermentation medium of the present invention, preferably an optimized fermentation medium.

In the two-phase fermentation method, the vitamin D3 is added in an amount of 0.6g/L to 1g/L calculated by the volume of the fermentation medium during inoculation in the step (1); the volume ratio of n-heptane to fermentation medium is 1:8-1:3 or 1:8-1:4, preferably 1:5. In the two-phase fermentation method, beta-cyclodextrin can be added or not; the beta-cyclodextrin may be dosed in the range of 3g/L to 8g/L, preferably 5g/L, calculated on the volume of fermentation medium at the time of inoculation in step (1).

In some embodiments, the two-phase fermentation process comprises:

(1) Inoculating spores of the autotrophic Pseudonocardia G-5H1 into a seed culture medium, and performing shaking culture at a constant temperature of 25-30 ℃ and 220-280 rpm for 48-72 hours, preferably 60 hours; then, the seed culture medium and the fermentation culture medium are transferred into the fermentation culture medium (such as the fermentation culture medium after optimization and improvement) according to the volume ratio of the seed culture medium to the fermentation culture medium, and the constant temperature shaking table culture is carried out for 3 to 7 days, preferably 5 days at the temperature of 25 ℃ to 30 ℃ and the constant temperature of 220rpm to 280 rpm;

(2) Adding vitamin D3 and n-heptane, optionally adding beta-cyclodextrin, and reacting for 2-6 days, preferably 4 days, to obtain the final product; post-treatment is carried out to obtain a target product; wherein, calculated by the volume of the fermentation medium when inoculated in the step (1), the vitamin D3 is added in an amount of 0.6g/L to 1g/L, the vitamin D3 is added after being dissolved by ethanol accounting for 0.1 to 2 percent of the volume of the fermentation medium, the volume ratio of n-heptane to the fermentation medium is 1:8 to 1:3, and the beta-cyclodextrin can be added in an amount of 0g/L or 3g/L to 8g/L, preferably 5g/L.

In some embodiments, the two-phase fermentation process comprises:

(1) Collecting spores of the autotrophic Pseudonocardia G-5H1 by using a glycerol aqueous solution with the mass ratio of 20% to obtain a spore suspension, and inoculating the spores into a seed culture medium according to the inoculum size with the volume ratio of 0.001% -1% and the preferred volume ratio of 0.1% and the volume ratio of 0.001% -1%, and culturing the spores in a constant-temperature shaking table at the temperature of 25-30 ℃ and the speed of 220-280 rpm for 48-72 hours, preferably 60 hours; then, according to the volume ratio of the seed culture medium to the fermentation culture medium, inoculating the seed culture medium into the fermentation culture medium (such as the optimized and improved fermentation culture medium) according to the inoculum size of 5-10% of the volume ratio, and carrying out shaking culture at constant temperature of 25-30 ℃ and 220-280 rpm for 3-7 days, preferably 5 days;

(2) Adding vitamin D3 and n-heptane, optionally adding beta-cyclodextrin, and reacting for 2-6 days, preferably 4 days, to obtain the final product; post-treatment is carried out to obtain a target product; wherein, the vitamin D3 is added in an amount of 0.6g/L to 1g/L calculated by the volume of the fermentation medium in the step (1), the vitamin D3 is dissolved by ethanol accounting for 0.1 percent to 2 percent, preferably 0.6 percent of the volume of the fermentation medium, the volume ratio of the n-heptane to the fermentation medium is 1:8 to 1:4, preferably 1:5, and the beta-cyclodextrin is 5g/L or 0g/L.

In some embodiments, the two-phase fermentation process comprises:

(1) The autotrophic Pseudonocardia G-5H1 is inoculated in a seed culture medium, and is cultured for 2 to 3 days at the temperature of 28 ℃ and the rpm of 250 rpm; then, the seed culture medium and the fermentation culture medium are transferred to the fermentation culture medium according to the inoculation amount of 10 percent of the volume ratio, and the seed culture medium and the fermentation culture medium are cultured for 5 days at 28 ℃ and 250 rpm;

(2) Adding vitamin D3 and n-heptane into the fermented fermentation medium, optionally adding beta-cyclodextrin, and reacting at 28 ℃ and 250rpm for 4 days to finish the reaction; post-treatment is carried out to obtain calcitriol and/or calcitriol; wherein, the vitamin D3 is added into the fermentation medium in the step (1) in a volume ratio of 0.6g/L to 1g/L, each 1g of vitamin D3 is dissolved by 15mL of ethanol, the volume ratio of n-heptane to the fermentation medium is 1:5, and the beta-cyclodextrin is 5g/L or 0g/L.

In some embodiments, the two-phase fermentation process comprises:

(1) Taking spores from a Gaoshi No. 1 plate with mature autotrophic Pseudonocardia G-5H1 spores, inoculating the spores into a seed culture medium, and culturing at 28 ℃ and 250rpm for 2-3 days; then transferring the fermentation medium according to the volume ratio of the seed medium to the fermentation medium and the inoculum size of 10 percent of the volume ratio, and culturing for 5 days at 28 ℃ and 250 rpm;

(2) Adding vitamin D3, n-heptane and beta-cyclodextrin into the fermented fermentation medium, and reacting at 28 ℃ and 250rpm for 4 days to finish the reaction; post-treatment is carried out to obtain calcitriol and/or calcitriol; wherein, calculated by the volume of the fermentation medium in the step (1), the vitamin D3 is added into the fermentation medium in a proportion of 0.6g/L to 1g/L, each 1g of vitamin D3 is dissolved by 15mL of ethanol, the beta-cyclodextrin is 5g/L, and the volume ratio of the n-heptane to the fermentation medium is 1:5.

The post-processing may include: adding extraction solvent such as ethyl acetate into the reaction system, separating liquid, collecting organic phase, removing solvent from the organic phase, and/or separating by column chromatography or other suitable method to obtain target product calcitriol and/or calcitriol; the eluent in the column chromatography can be ethyl acetate and petroleum ether, and the volume ratio can be 30:70.

After the reaction is finished, part of the reaction liquid can be taken, a shaking bottle is processed for 5min by using 90% power ultrasonic treatment, 5ml of the reaction liquid is taken, 5ml of absolute ethyl alcohol is added, and after uniform mixing, the mixture is centrifuged for 2min at 12000rpm; the supernatant was filtered and analyzed for product yield by HPLC. The inventor finds that the yield of the calcitonin can reach more than 500mg/L, which is improved by about 5 times compared with the original yield; the yield of calcitriol can reach about 108mg/L, and is improved by 10 times compared with the original yield.

The invention adopts a two-phase fermentation method of a two-phase reaction system, which can shorten the fermentation time, and improve the fermentation yield of calcitriol and/or calcitriol by 5 times or more compared with the original yield, and the yield can reach 500mg/L or more.

In the description of the present specification, the terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

In the present invention, the expressions such as "Pseudonocardia autotrophica G-5H1" and "G-5H1" mean the same strain; VD3 represents vitamin D3 and PBS represents phosphate buffered saline.

In the invention, mmol represents millimoles, h represents hours, g represents grams, mL represents milliliters, and mu L represents microliters; when referring to time, d represents day, h represents hour, min represents minute, and s represents second; rpm indicates revolutions per minute.

In the present invention, the original strain means a wild-type strain such as: the preservation number is CGMCC 4.1211, and the name is Pseudonocardia autotrophica. The high-yield strain refers to: the original strain is subjected to ultraviolet mutagenesis and ribosome engineering mutagenesis based strain evolution, and under the same conditions, when the original strain is used for preparing the same target product with a substrate, the yield of the original strain is improved compared with that of the original strain.

In the invention, the yield calculation method comprises the following steps: yield = conversion x amount of substrate or substrate concentration; if the conversion rate is 90%, and the concentration of the added substrate vitamin D3 is 0.6g/L, the yield is 90%. Times.0.6=0.54 g/L; alternatively, the conversion was 90%, and the yield was 90%. Times.0.6=0.54 g when 0.6g of vitamin D3 was added as a substrate to 1L of fermentation medium.

In the present invention, HPLC analysis is described in volume 285, 41, pages 31193-31201 (JOURNAL OF BIOLOGICAL CHEMISTRY, VOL 285,NO 41,PP 31193-31201).

In the invention, the seed culture medium comprises the following components in percentage by weight: 15g/L of soybean peptone, 15g/L of glucose, 5g/L of sodium chloride, 2g/L of calcium carbonate, pH value controlled to be 7.0 and the balance of water.

In the invention, the culture medium of Gaoshi No. 1 comprises the following components in percentage by weight: soluble starch 20g/L, agar 20g/L,FeSO₄·7H₂O 0.01g/L,MgSO₄·7H₂O 0.5g/L,K₂HPO₄·3H₂O 0.5g/L,KNO₃ 1g/L,NaCl 0.5g/L,pH value 7.4-7.6.

In the present invention, a component of the fermentation medium or a component used for preparing the fermentation medium may include or be a hydrate thereof, and the concentration of the component is calculated as an anhydrate; feSO ₄ may be used, as may FeSO ₄·7H₂ O, when the formulation components contain FeSO ₄, and the concentration thereof may be calculated as the mass (g) of FeSO ₄ to the total volume (L) of the fermentation medium. In order to achieve the desired pH of the fermentation medium, an acid or base such as hydrochloric acid or potassium hydroxide may be used to adjust the pH.

In the present invention, when numerical values are referred to, whether "about" or "approximately" are used, the numerical values may differ by no more than + -10%, i.e., the error may be-10%, + -8%, + -5%, + -2%, + -1%, or + -0.5%, etc.

Detailed Description

In order to better understand the technical solution of the present invention, the following further discloses some non-limiting examples, which are further described in detail.

The reagents used in the present invention are all commercially available or can be prepared by the methods described herein.

Experiment 1:

Spore suspension was obtained: after growing on a solid inclined plane of Gaoshan No. 1 at 28 ℃ for 7 days, collecting spores by using a sterile 20% glycerol water solution (mass ratio), filling the received glycerol water into a sterile 250mL triangular bottle containing glass beads, plugging a silica gel plug, and placing the triangular bottle in a shaking table to oscillate for 30min at 28 ℃ and 220 rpm; filtering with two layers of parchment paper, collecting filtrate, mixing, packaging into sterile strain preservation tube, 1 mL/tube, and preserving at-80deg.C; a spore suspension was obtained.

Bacterial count: plate colony counting was used; first atPouring 20mL of Gaoshi No. 1 culture medium into a sterile culture dish, and manufacturing a flat plate; secondly, taking a tube 1 in the strain preservation tube for diluting the spore suspension, taking 100 mu L of the spore suspension from the tube 1, adding 900 mu L of sterile water into the spore suspension, namely diluting the spore suspension to 10 ^-1, and carrying out gradient dilution to 10 ^-6, & gt to 10 ^-9; the spores were collected by plating 3 plates with 10 ^-6～10^-9 concentrations of each of the inoculations, culturing at 30℃for 7 days at an inoculum size of 100. Mu.L/block, and then counting and averaging. Dilution with sterile water or aqueous glycerol yields spore suspensions with spore concentrations of 10 ⁶/mL or 10 ⁹/mL.

Experiment 2: ultraviolet mutagenesis of autotrophic Pseudonocardia (collection number CGMCC 4.1211, name: pseudonocardia autotrophica)

Taking 5mL spore suspension with spore concentration of 106/mL, adding into aseptic containing radix et rhizoma Rhei needleIn the dish, the medium was irradiated under an ultraviolet lamp (UV 254, 19W) at 30cm for 15s, 30s, 45s, 60s, 75s, 90s, respectively, with stirring. Inoculating 3 blocks with each irradiated spore suspensionThe inoculum size of the Gaoshi No. 1 plate was 100. Mu.L/block, and the culture was performed in a dark room at a constant temperature of 28℃for 5 days, followed by counting, and the mortality was calculated.

Results: the irradiation time at a mortality rate of not less than 99.99% was 60s.

Experiment 3: ribosome engineering mutagenesis of autotrophic Pseudonocardia (collection number CGMCC 4.1211, name: pseudonocardia autotrophica)

Precisely weighing three antibiotics of streptomycin (code number SS), rifamycin (code number Rif) and gentamicin (code number Gen), dissolving SS and Gen with pure water, dissolving Rif with dimethyl sulfoxide, and then filtering with a sterile disposable needle-type filter of 0.22 mu m to prepare sterile solutions of three antibiotics with concentration of 100 mg/ml; subpackaging into 1 mL/tube, and preserving at-20deg.C for use. Preparing a culture medium of high-grade No. 1, sterilizing at 121 ℃ for 20min, and cooling to about 50 ℃; according to the antibiotic concentrations in Table 1, 3 plates of Gaoshi No. 1 medium each containing three antibiotics at each concentration were prepared.

Inoculating the Gaoshi No. 1 culture medium plate with spore suspension having spore concentration of 106 pieces/mL, wherein the inoculation amount is 100 mu L/block; after incubation at 28℃for 5-7 days, counts were taken to determine the Minimum Inhibitory Concentration (MIC) of the different antibiotics, respectively.

Results: the MIC of Gen was determined to be 5mg/L, the MIC of SS was determined to be 5mg/L, and the MIC of Rif was determined to be 0.1mg/L.

Table 1: antibiotic concentration

Antibiotics	Concentration of	Concentration of	Concentration of	Concentration of	Concentration of
						Gen	2.5mg/L	5mg/L	10mg/L	50mg/L	100mg/L
SS	2.5mg/L	5mg/L	10mg/L	50mg/L	100mg/L
						Rif	0.05mg/L	0.1mg/L	1mg/L	10mg/L	20mg/L

Example 1 combination of ultraviolet mutagenesis and ribosome engineering mutagenesis of Pseudonocardia aeroponica (collection number CGMCC 4.1211, name: pseudonocardia autotrophica) and screening

According to the method of experiment 1, spore suspension of autotrophic Pseudonocardia (preservation number CGMCC 4.1211, name: pseudonocardia autotrophica) is obtained; adding 5mL spore suspension with spore concentration of 10 ⁹/mL into aseptic containing radix et rhizoma Rhei needleIn the dish, the dishes were irradiated under ultraviolet lamp (UV 254, 19W) at 30cm with stirring for 60s, respectively. With the spore suspension after irradiation, 100. Mu.L/block were inoculated onto plates 1 Gao's containing antibiotics at the respective concentrations (1-fold MIC, 5mg/L for Gen, 5mg/L for SS, 0.1mg/L for Rif), 20 plates were inoculated in parallel for each antibiotic concentration, 10 plates without antibiotics were also inoculated (as a reference group), and a total of 70 plates were incubated in a dark room at 28℃for 5-7 days; all surviving single colonies were then removed; the results are shown in Table 2.

All surviving single colonies were inoculated into 300 μl of medium/well 96-well plate medium No. 1, incubated at 28 ℃ for 7 days; then, transfer to 300 u L medium/96 hole plate primary fermentation medium, control each hole 240 u L medium, 28 degrees, 500rpm micro-pore plate shaking culture for 5 days; then centrifugally collecting thalli by a micro-pore plate centrifuge, and washing for 2 times by 10mmol/L PBS (phosphate buffered saline), wherein 200 mu L of thalli are used each time; then 60 mu L of 10mmol/L veratrole is added to each well, 120 mu L of PBS is added, after 1h of reaction at 28 ℃,1 mu L of 0.08 g/L2, 4 dinitrophenylhydrazine is added for color development, and the reaction is carried out overnight at 28 ℃ (12 h-16 h). After the reaction is finished, selecting colonies with colors changed from yellow to orange or deepened, and obtaining potential high-yield strains; the results are shown in Table 2.

Shake flask fermentation was performed on a total of 186 potential high-yielding strains (darkened colonies): inoculating single colonies of the potential high-yield strain into a seed culture medium respectively, and carrying out shaking culture at a constant temperature of 28 ℃ and 250rpm for 3 days; then transferring the inoculated amount of 10 percent (volume ratio of the seed culture medium to the fermentation culture medium) of the volume ratio into the optimized fermentation culture medium, and carrying out shaking culture at the constant temperature of 250rpm at the temperature of 28 ℃ for 5 days. Then adding substrate vitamin D3,1g of vitamin D3 into the fermentation medium, using absolute ethyl alcohol to fix the volume to 15mL,5g of beta-cyclodextrin, and using 40mL of water for dissolution; 600. Mu.L of vitamin D3 ethanol solution, 2mL of beta-cyclodextrin aqueous solution and 10mL of n-heptane are added to each 50mL of fermentation medium; after culturing at 28℃for 4 days at 250rpm, the samples were collected. Sampling, performing 90% power ultrasonic treatment for 5min, taking 5mL of fermentation liquor, adding 5mL of absolute ethyl alcohol, uniformly mixing, and centrifuging for 2min at 12000rpm; the supernatant was filtered and analyzed by HPLC. Screening out strains with higher yields of calcitriol and calcitriol, namely high-yield strains; the results are shown in tables 2 and 3.

Results: a total of 192 single colonies were selected in all the reference groups, wherein 25 strains of the number of the darkened colonies were larger than 0 strains of the original bacteria; gen group picked 72 single colonies at 1-fold MIC (5 mg/L) concentration, 5 strains of darkened colonies; the SS group picked 576 single colonies at 1-fold MIC (5 mg/L) concentration, 156 colonies with darkened color; no viable colonies were found in the Rif group; see table 2. According to HPLC detection results, the reference group and the Gen group, the Rif group has no strains with improved yield, and the SS group has 5 strains; the 5 mutant strains including G-5B5, G-5B8, G-5B10, G-5C1 and G-5H1, which had the highest yields, were selected, and the yields were significantly improved over the original strains, wherein the shake flask-fermented calcitol yield of G-5H1 reached 525mg/L, and the yields were improved by nearly 5-fold as compared to the yields in the original strains, see tables 2 and 3.

Table 2: ultraviolet and ribosome engineering mutagenesis and screening results of original strain are summarized

Colony/antibiotic and concentration	Reference group	Gen 5mg/L	SS 5mg/L	Rif 0.1mg/L
					Number of single colonies	192	72	576	0
Number of colonies darkened	25	5	156	0
					Yield enhancement	0	0	5	0

Table 3: comparison of the high-yield Strain with the calcitol yield obtained from the original Strain

Strain

Original strain

G-5B5

G-5B8

G-5B10

G-5C1

G-5H1

Yield of products

102mg/L

488mg/L

515mg/L

356mg/L

484mg/L

525mg/L

Example 2: optimization and improvement of fermentation culture medium

Based on the original fermentation culture medium of the autotrophic Pseudonocardia (preservation number CGMCC4.1211, name: pseudonocardia autotrophica), the optimization and improvement are carried out.

For optimization and improvement of the carbon source, glucose, fructose, mannitol, glycerol, maltose, dextrin, corn flour, corn steep liquor, soluble starch, galactose, lactose, sorbitol, xylose and sucrose were used as initial carbon sources, respectively, and the conversion of calcitol under each condition was evaluated. The result shows that the conversion rate can be better improved when the sucrose and the glycerol are used as single carbon sources, the price of the glycerol is about 2 times of that of the sucrose, the sucrose is used as the first quick-acting carbon source of the fermentation medium, and the corn steep liquor is determined as the second long-acting carbon source through multiple optimization; for seed engagement, it is advantageous to add 5g/L glucose; after a series of test comparisons, a combination of 10g/L sucrose, 5g/L glucose and 15.37g/L corn steep liquor was determined as the preferred combination.

After the composition of the carbon source was determined, 14 common nitrogen sources including peptone, tryptone, yeast powder, soybean peptone, malt extract, beef extract, enzymatic casein, corn steep liquor, corn flour, soybean meal, cotton seed meal, soluble fish meal, L-tyrosine and plant peptone (Phytone, BD company) were compared in order to optimize the nitrogen source. The comparison result shows that the tryptone, the yeast powder, the soybean peptone and the soybean cake powder can well improve the conversion rate; the multielement combination optimization shows that the combination of yeast powder and soybean peptone is obviously superior to other compound combinations in the aspects of potency, production cost and the like; after a series of test comparisons, a combination of 35g/L of soybean peptone and 31.5g/L of yeast powder was determined as a preferred combination.

In order to optimize inorganic salts in the culture medium, the inventor carries out comparative tests on the inclusion NaCl、KCl、MgSO₄、FeSO₄、CaCl₂、MnCl₂、(NH₄)₂SO₄、K₂HPO₄、NaF, and the results show that the addition of MgSO ₄ and FeSO ₄ can significantly improve the conversion rate of the calcitol (this does not exclude that the enzymes involved in conversion need Mg ²⁺ and Fe ²⁺), phosphate can promote the growth of thalli, and NaCl plays an important role in the electron transfer of a respiratory chain; a certain amount of CaCO ₃ can balance the pH value of the fermentation liquor and is beneficial to the growth of mycelium; finally, the preferred combination of inorganic salts is FeSO ₄ 0.1g/L、MgSO₄ 0.5g/L、CaCO₃2g/L、NaCl 5g/L、K₂HPO₄ 0.1.1 g/L and NaF 0.1g/L.

Considering the extremely low solubility of the substrate and the product, the cosolvent, the surfactant and the reagent for promoting the formation of the membrane channel which may be added in the culture medium are screened, and the test comparison is carried out on tween 20, tween 80, triton X-100, mannitol, sorbitol, betaine, PEG4000, nisin, sodium dodecyl sulfate, caCl ₂, beta-cyclodextrin, hydroxypropyl-cyclodextrin and sulfobutyl-cyclodextrin, and the test result shows that the effects of the surfactant tween 20, tween 80 and sorbitol are obvious, and the cosolvent beta-cyclodextrin is relatively good. The multi-element combination optimization experiment shows that the combination of 5ml/L Tween 20 and 5g/L beta-cyclodextrin is obviously superior to other combinations.

After multiple tests and combination test comparison, the most preferred fermentation medium, components or components used for preparation and the concentration thereof are finally determined according to the experimental result: 10g/L of sucrose, 5g/L of glucose, 15.37g/L of corn steep liquor, 35g/L of soybean peptone, 31.5g/L,FeSO₄ 0.1g/L,MgSO₄ 0.5g/L,CaCO₃ 2g/L,NaCl 5g/L,K₂HPO₄ 0.1g/L,NaF 0.1g/L ml/L of yeast powder and 5ml/L of tween 20, wherein the pH value is 7.0, the beta-cyclodextrin is 0g/L or 5g/L, and the balance is water.

For example carbon nitrogen source optimization: a4-factor experiment (see tables 4 and 5, referred to in units of g/L) of carbon and nitrogen sources was designed using the Design expert 8.0.6 software optimization, and fermentation media were prepared according to the Design, 3 in parallel per group. 1 loop of spores are scraped from a Gao's No. 1 plate with mature spores of autotrophic Pseudonocardia (preservation number CGMCC4.1211, english name: pseudonocardia sp) to inoculate a seed culture medium, and the culture is carried out at 28 ℃ and 220rpm for 3d. The fermentation medium was inoculated at an inoculum size of 10%,28℃and 250rpm for 5 days. Weighing VD 31 g, and using absolute ethyl alcohol to fix the volume to 15mL; 5g of beta-cyclodextrin is weighed and dissolved by 40mL of water; 225 mu L of VD3 solution, 2mL of beta-cyclodextrin aqueous solution and 10mL of n-heptane are added into each 50mL of fermentation medium; culturing was continued at 28℃and 250rpm for 4d, after which the samples were collected.

Shaking the flask for 5min by 90% power ultrasonic treatment, adding 5mL of absolute ethyl alcohol into 5mL of fermentation liquid, uniformly mixing, and centrifuging for 2min at 12000rpm. The supernatant was filtered and analyzed by HPLC to calculate the average conversion for each group, which was filled into the experimental design of the software (see table 4), and the P value was calculated according to the software, with factors of P <0.05 being significant factors. Finally, the following steps are: a preferred combination of 10g/L sucrose, 15.37g/L corn steep liquor, 35g/L soybean peptone and 31.5g/L yeast powder (see Table 6, referred to in g/L).

Table 4: carbon-nitrogen source response surface experimental design combination and result

Table 5: carbon-nitrogen source response surface experimental design

Table 6: carbon nitrogen source response surface experiment

Example 3: determination of fermentation, reaction time and reaction System

Both the substrate vitamin D3 and the product calcitol are fat-soluble and inhibit the growth of microorganisms, so that separation of the cell, substrate and product, minimizing contact, reduces toxicity and is beneficial to product formation.

Different fermentation time and reaction time are examined, the fermentation is carried out for 5 days, the reaction is carried out for 4 days, the yield of the target product is relatively highest, and the preferable combination mode is determined to be fermentation for 5 days and reaction for 4 days.

The reaction system is examined, and the reaction system of n-hexane, n-heptane, cyclohexane, ethanol, ethyl acetate, isopropanol, soybean oil, peanut oil or corn oil and the fermentation medium is compared, so that the result shows that the reaction system of n-heptane and the fermentation medium is more favorable for generating target products, and the conversion rate is obviously improved compared with other combinations when the n-heptane is the fermentation medium=1:5 (V/V).

Example 4: preparation of calcitol

Scraping spores (about 1cm multiplied by 1cm in size) from a Gao's No. 1 plate in which the spores of the autotrophic Pseudonocardia G-5H1 are mature, inoculating the spores into a seed culture medium, and culturing at 28 ℃ and 220rpm for 3d; the most preferable fermentation medium is inoculated, the inoculation amount is 10%, the temperature is 28 ℃, and the culture and the fermentation are carried out at 250rpm for 5 days.

1G of vitamin D3 is weighed, absolute ethyl alcohol is used for fixing the volume to 15mL, 5g of beta-cyclodextrin is weighed, 40mL of water is used for dissolving, 600 mu L of vitamin D3 (VD 3) solution, 2mL of beta-cyclodextrin solution and 10mL of n-heptane are added into each 50mL of fermentation medium; culturing at 28deg.C at 250rpm for 4 days, and collecting sample; ethyl acetate, optionally filtering, separating, collecting organic phase, evaporating under reduced pressure to obtain mixture mainly comprising calcitriol and calcitriol, and subjecting the mixture to silica gel column chromatography with eluent ethyl acetate: petroleum ether=3:7 (volume ratio).

Sampling the reaction fermentation liquid after the reaction is finished, performing 90% power ultrasonic treatment on the reaction fermentation liquid for 5min, adding 5mL of absolute ethyl alcohol into each 5mL of fermentation liquid, uniformly mixing, and centrifuging for 2min at 12000rpm; the supernatant is filtered and then is analyzed by HPLC, and the result shows that the yield of the calcitonin reaches about 525mg/L, which is improved by about 5 times compared with the original yield; the calcitriol yield reaches about 108mg/L, and is improved by about 10 times compared with the original yield.

By adopting the technical process, different strains, culture mediums and/or fermentation and reaction time are used, and the obtained calcitonin has the following yield:

Bacterial strain, culture medium	Fermentation, reaction time	Ossified diol yield
			Original strain, original fermentation medium	Fermenting for 7 days, reacting for 5 days	110mg/L-90mg/L
G-5H1 and other high-yield strains and original fermentation culture medium	Fermenting for 5 days, reacting for 4 days	300mg/L-400mg/L
			G-5H1 and other high-yield strains and optimized fermentation culture medium	Fermenting for 5 days, reacting for 4 days	>500mg/L

According to the results, the yield is obviously improved by adopting the high-yield strain, and the yield is obviously improved by adopting the high-yield strain and the optimized fermentation medium.

While the methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and combinations of the methods and applications described herein can be made and applied within the spirit and scope of the invention. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included within the present invention.

Claims

1. An autotrophic nocardia (Pseudonocardia autotrophica) G-5H1, characterized in that: the preservation number is CGMCC No. 19461, the preservation number is China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms in Beijing, the preservation date is 09 in 2020 and the preservation address is North Star Xiyu No. 1 and 3 in the Korean region of Beijing.

2. Use of the autotrophic nocardia G-5H1 of claim 1 for the preparation of calcitriol and/or calcitriol.

3. A process for the preparation of calcitriol and/or calcitriol comprising the use of the autotrophic nocardia G-5H1 of claim 1 by a two-phase fermentation process; wherein the two-phase fermentation method comprises the following steps:

(1) Inoculating the autotrophic Pseudonocardia G-5H1 of claim 1 into a seed culture medium, shake culturing, inoculating into a fermentation culture medium, and shake culturing;

(2) Adding vitamin D ₃ and n-heptane into the culture solution obtained in the step (1), and adding beta-cyclodextrin to react; post-treatment is carried out after the reaction is finished to obtain calcitriol and/or calcitriol; the dosage of vitamin D ₃ is 0.6g/L-1g/L and the volume ratio of n-heptane to the fermentation medium is 1:8-1:3 calculated by the volume of the fermentation medium in the step (1).

4. The method of claim 3, wherein the two-phase fermentation process comprises:

(1) Inoculating spores of the autotrophic Pseudonocardia G-5H1 into a seed culture medium, and shake culturing at 25-30 ℃ and 220-280 rpm for 48-72 hours; then, the seed culture medium and the fermentation culture medium are used for transferring and planting in the fermentation culture medium according to the inoculum size of 5-10 percent of the volume ratio, and the constant temperature shaking table culture is carried out for 3-7 days at the temperature of 25-30 ℃ and the constant temperature of 220-280 rpm;

(2) Adding vitamin D ₃ and n-heptane into the culture solution obtained in the step (1), adding beta-cyclodextrin, and reacting for 2-6 days, wherein the reaction is finished; adding extraction solvent, separating, collecting organic phase, removing solvent and/or separating by column chromatography to obtain calcitriol and/or calcitriol; wherein, the vitamin D ₃ is added after being dissolved by ethanol, and the dosage of the ethanol is 0.1-2% of the volume of the fermentation medium when the fermentation medium is inoculated in the step (1), and the beta-cyclodextrin is 3 g/L-8 g/L.

5. The method of claim 3, wherein the two-phase fermentation process comprises:

(1) Inoculating the autotrophic Pseudonocardia G-5H1 into a seed culture medium, and culturing at 28 ℃ and 250rpm for 48-72 hours; then transferring the seed into a fermentation culture medium according to the inoculum size of 10 percent of the volume ratio, and culturing for 5 days at 28 ℃ and 250 rpm;

(2) Adding vitamin D ₃ and n-heptane into the culture solution obtained in the step (1), adding beta-cyclodextrin, and reacting at 28 ℃ and 250rpm for 4 days, wherein the reaction is finished; post-treatment is carried out to obtain calcitriol and/or calcitriol; wherein, calculated by the volume of the fermentation medium in the step (1), the dosage of the vitamin D ₃ is 0.6g/L-1g/L, each 1g of vitamin D ₃ is dissolved by 15mL of ethanol, the volume ratio of the n-heptane to the fermentation medium is 1:5, and the beta-cyclodextrin is 5g/L.

6. The method according to any one of claims 3 to 5, wherein the concentration of the components of the fermentation medium or the components used in the formulation is, based on the total volume: 10g/L of sucrose, 5g/L of glucose, 15.37g/L of corn steep liquor, 35g/L of soybean peptone, and 31.5g/L,FeSO₄ 0.1g/L,MgSO₄ 0.5g/L,CaCO₃2g/L,NaCl 5g/L,K₂HPO₄0.1g/L,NaF 0.1g/L, ml of yeast powder Tween 20, wherein the pH value is 7.0.