Method for producing 11 alpha-hydroxy canrenone by efficiently converting canrenone

Abstract

The invention discloses a method for producing 11 alpha-hydroxycanrenone by efficiently converting canrenone, which comprises the following steps: inoculating recombinant pichia pastoris into a shake flask filled with a seed culture medium, and culturing until the OD600 value of a bacterial solution reaches 8-10 to obtain a seed solution; inoculating the seed solution into a fermentation tank filled with a fermentation culture medium according to the inoculum size of 3-8%, and carrying out fermentation culture; the fermentation culture medium contains inositol; carrying out fermentation culture until dissolved oxygen rises again, and starting to add methanol in a fed-batch manner to carry out induced expression; and then adding canrenone into the fermentation tank, and ending fermentation until the canrenone conversion rate reaches 90% or above. Recombinant pichia pastoris microorganisms are adopted to convert canrenone to prepare 11 alpha-hydroxy canrenone, meanwhile, components of a fermentation culture medium are optimized, the production cycle can be greatly shortened, particularly, the effect of shortening the production cycle with high feeding concentration is remarkable and is obviously lower than that in the prior art, and therefore the production efficiency is greatly improved, and the production cost is reduced. And the time cost is reduced.

Description

Technical Field

[0001] This invention belongs to the field of microbial transformation technology, specifically relating to a method for efficiently converting canrone to produce 11α-hydroxycanrone. Background Technology

[0002] 11α-Hydroxycanrenone (CAS No. 192569-17-8) is a steroidal derivative formed by the C11 hydroxylation of canrenone and a key pharmaceutical intermediate in the preparation of eplerenone. Eplerenone is a commonly used antihypertensive drug with proven efficacy in treating heart failure and myocardial infarction. Compared with other cardiovascular drugs, it has fewer adverse reactions and is better tolerable. 11α-Hydroxycanrenone can also be used to prepare a series of useful canrenone derivatives, and therefore has attracted considerable attention.

[0003] The preparation of 1,1-α-hydroxycanrenone can be achieved through chemical synthesis and microbial transformation. However, chemical synthesis is characterized by numerous reaction steps, low specificity, abundant byproducts, and significant environmental pollution. Therefore, an increasing number of studies have opted for the highly specific and environmentally friendly microbial transformation method to prepare 1,1-α-hydroxycanrenone.

[0004] Currently, the main microorganisms used to convert canrenone into 11α-hydroxycanrenone include Aspergillus ochraceus and rhizozymes. Among them, Aspergillus ochraceus is the most widely used in industrial applications. However, the method of using Aspergillus ochraceus for microbial conversion to prepare 11α-hydroxycanrenone has problems such as long production cycle, low feed concentration, and high cost, which seriously restricts the large-scale production of 11α-hydroxycanrenone.

[0005] For example, Chinese patent documents CN1727494A, CN103255192A, CN116970674A, and CN118256588A all have a feed concentration of no more than 20 g / L, and the conversion time alone is more than 60 hours (the higher the feed concentration, the longer the conversion time). The entire production cycle is more than 100 hours. CN118256588A also has the problem of using expensive β-glucosidase, which leads to higher production costs.

[0006] Chinese patent document CN110713941A optimizes the microbial transformation of Aspergillus ochraceus by directional modification of Aspergillus ochraceus strains. However, for a feed concentration of 20 g / L, it still requires more than 60 hours of transformation to obtain a transformation rate of over 90%.

[0007] Chinese patent document CN112980910A optimizes the microbial transformation of Aspergillus ochraceus using a phase transfer catalyst. At a feed concentration of 30 g / L, a conversion rate of 95.5% can be achieved in just 54 hours. However, at a feed concentration of 40 g / L, the conversion rate is only 76.8–80.3% after 60 hours, which is still not ideal. Furthermore, the phase transfer catalyst exhibits significant cytotoxicity and is difficult to process, potentially leading to catalyst residue and affecting product quality and safety.

[0008] Currently, there are literature reports on the use of Pichia pastoris or recombinant Pichia pastoris for C11α-hydroxylation.

[0009] For example, the recombinant Pichia pastoris constructed in Reference 1 can efficiently convert L-ethylsterene diketone to 11α-L-ethylsterene diketone, but it does not mention canlione or other substrates.

[0010] For example, the recombinant Pichia pastoris constructed by Chinese patent document CN117660216A can efficiently convert 4-androstenedione to produce 11α-hydroxy4-androstenedione, but it also does not mention canlione or other substrates.

[0011] For example, the recombinant Pichia pastoris constructed by Chinese patent document CN118853605A can efficiently convert 18-methylnandrolone, 17-hydroxyprogesterone, 18-methyldiketone, progesterone, etc. to produce the corresponding 11α-hydroxy derivatives. However, for canrenone, the conversion rate is only 80.5% after 72 hours, which still cannot solve the problem of efficiently converting canrenone to produce 11α-hydroxycanrenone.

[0012] Reference 1: “Expression and functional analysis of the ochratoxin steroid 11α-hydroxylase gene AOH in Pichia pastoris”, Guo Kai et al., Journal of Tianjin University of Science and Technology, Vol. 32, No. 5, pp. 34-38, 78, October 2017. Summary of the Invention

[0013] The purpose of this invention is to solve the above-mentioned problems and provide a method for the efficient conversion of canrone to 11α-hydroxycanrone with a shorter production cycle, especially with a shorter production cycle under high feed concentration.

[0014] The technical solution to achieve the objective of this invention is: a method for efficiently converting canrenone to produce 11α-hydroxycanrenone, characterized by the following steps: ① Seed culture: Recombinant Pichia pastoris was inoculated into a shake flask containing seed culture medium and cultured until the OD of the bacterial culture reached a certain level. 600 When the value reaches 8-10, seed solution is obtained.

[0015] The recombinant Pichia pastoris was constructed by introducing the hydroxylase gene from Aspergillus ochraceus into the host Pichia pastoris GS115 using gene cloning technology, as detailed in Reference 1.

[0016] ② Fermentation tank inoculation: The seed liquid obtained in step ① is inoculated into a fermentation tank containing fermentation culture medium at an inoculation rate of 3-8% for fermentation culture; the fermentation culture medium contains inositol.

[0017] ③ Induction of expression and substrate addition: The dissolved oxygen in the fermenter first decreases and then rises (as the bacteria consume nutrients) (as nutrients are gradually depleted). After the dissolved oxygen rises to above 60%, methanol is added to induce expression. After 1.5 to 3 hours of induction, canrone is added to the fermenter until the canrone conversion rate reaches above 90%. Fermentation is then stopped, and the fermentation broth is collected for subsequent extraction and purification to obtain 11α-hydroxycanrone.

[0018] In step ① above, the seed culture medium used is YPD medium, with the following formula: yeast extract 10g / L, peptone 20g / L, and glucose 20g / L. YPD medium can provide sufficient nutrition for the rapid growth of recombinant Pichia pastoris seeds, ensuring seed viability and quantity.

[0019] In step ① above, the seed culture temperature is 28-32℃, the rotation speed is 200-250rpm, and the time is 20-24h.

[0020] In step ② above, the fermentation medium comprises: glucose 25–35 g / L, corn steep liquor powder 15–25 g / L, potassium dihydrogen phosphate 10–15 g / L, dipotassium hydrogen phosphate trihydrate 1.0–2.0 g / L, ammonium sulfate 1.5–2.5 g / L, magnesium sulfate 0.3–0.7 g / L, and inositol 0.01–0.05 g / L. Inositol is a key nutrient; the applicant has found through extensive experiments that it can significantly increase the hydroxylase activity of recombinant Pichia pastoris, thereby significantly improving conversion efficiency.

[0021] In step ② above, the initial conditions for fermentation culture are: temperature 28-32℃, pH value 5.0-5.5, rotation speed 500-1000rpm, ventilation rate 1-2L / min, and dissolved oxygen controlled above 20%.

[0022] In step ③ above, the initial flow rate of methanol is 1-2 mL / h·L, and the maximum flow rate of methanol is 5-6 mL / L·h.

[0023] In step ③ above, the concentration of canrone is 20–40 g / L.

[0024] The positive effects of this invention are: (1) The present invention uses recombinant Pichia pastoris microorganisms to transform canrone to prepare 11α-hydroxycanrone, and optimizes the fermentation medium components (adding inositol), which can greatly reduce the production cycle, especially for high feed concentrations, the production cycle is significantly shortened (from seed culture to the end of fermentation, the total time is only 60-70h), which is significantly lower than the existing technology, thereby greatly improving production efficiency and reducing time costs.

[0025] (2) Compared with other processes with high feed concentration (e.g., literature CN112980910A), the method of the present invention not only has the advantages of a shorter production cycle, but also has the advantages of low cost and controllable product quality and safety.

[0026] (3) The recombinant Pichia pastoris culture conditions of the present invention are mild and the fermentation process is easy to control, making it suitable for large-scale industrial production and effectively solving the bottleneck of large-scale production of traditional processes. Detailed Implementation

[0027] (Example 1) The specific method for efficiently converting canrenone to 11α-hydroxycanrenone in this embodiment is as follows: ① Seed culture: Prepare YPD medium (10g / L yeast extract, 20g / L peptone, 20g / L glucose), sterilize at 115℃ for 20min, and cool before use.

[0028] 0.2 mL of bacterial culture was taken from the recombinant Pichia pastoris glycerol cryopreservation tube and inoculated into a 500 mL shake flask containing 100 mL of YPD seed culture medium. The culture was incubated at 30 °C and 200 rpm for 22 h. The OD600 value of the bacterial culture was measured to be around 9, thus obtaining a highly viable seed culture.

[0029] ② Fermentation tank inoculation: Prepare fermentation culture medium (30g / L glucose, 20g / L corn steep liquor powder, 12.7g / L potassium dihydrogen phosphate, 1.4g / L dipotassium hydrogen phosphate trihydrate, 2g / L ammonium sulfate, 0.5g / L magnesium sulfate, 0.02g / L inositol), sterilize at 121℃ for 30min, and sterilize glucose separately at 115℃ for 20min.

[0030] The pH of the sterilized fermentation medium was adjusted to 5.0 with ammonia water. Then, the seed liquid obtained in step ① was inoculated into a 3L fermenter containing 1.5L of fermentation medium at a 5% inoculation rate. The initial fermentation conditions were controlled as follows: temperature 30℃, pH 5.0, rotation speed 800rpm, ventilation rate 1.5L / min, and dissolved oxygen controlled above 20%.

[0031] ③ Induction of expression and substrate addition: After 15-16 hours of fermentation, dissolved oxygen began to rise. When dissolved oxygen rose to above 60%, methanol was added initially at a rate of 2 ml / h·L. Dissolved oxygen was maintained above 20%, and the methanol flow rate was gradually increased, with a maximum flow rate of 6 ml / h·L. After 2 hours of induction of expression, canrone was added to the fermenter to a concentration of 30 g / L, and fermentation continued.

[0032] Results: After 40 hours of fermentation, the canrone conversion rate reached 90.3%. After fermentation was stopped and the fermentation broth was purified, the first-pass yield of 11α-hydroxycanrone was 83.6%.

[0033] The total production cycle (from seed culture to the end of fermentation) is 62 hours.

[0034] (Examples 2 to 4) The methods of each embodiment are basically the same as those of Embodiment 1, except that the amount of each component of the fermentation medium in step ② and the concentration of the feed in step ③ are shown in Table 1. The results are also shown in Table 1.

[0035] Table 1 Example 1 Example 2 Example 3 Example 4 glucose 30g / L 27g / L 33g / L 30g / L Corn liquor dry powder 20g / L 18g / L 22g / L 20g / L Potassium dihydrogen phosphate 12.7g / L 12.1g / L 13.3g / L 13.5g / L Dipotassium hydrogen phosphate trihydrate 1.4g / L 1.3g / L 1.5g / L 1.5g / L ammonium sulfate 2.0g / L 2.1g / L 2.0g / L 2.1g / L Magnesium sulfate 0.5g / L 0.5g / L 0.5g / L 0.5g / L Inositol 0.02g / L 0.02g / L 0.03g / L 0.03g / L Feed concentration 30g / L 20g / L 40g / L 35g / L End of fermentation time 40h 36h 45h 42h canridine conversion rate 90.3% 91.2% 90.1% 90.7% First-time yield 83.6% 82.7% 81.3% 80.8% Total production cycle 62h 58h 67h 64h

[0036] (Comparative Example 1) This comparative example is basically the same as Example 1, except that the fermentation medium does not contain inositol.

[0037] Results: After fermentation for 40 hours, the conversion rate of canrone was 82.9%, and the first-pass yield of the purified 11α-hydroxycanrone was 74.7%. Compared with Example 1, both the conversion rate and the first-pass yield decreased by about 10%.

[0038] This shows that inositol is crucial for maintaining the hydroxylase activity of recombinant Pichia pastoris, improving conversion efficiency, and increasing product yield.

[0039] (Comparative Example 2) This comparative example is basically the same as Example 1, except that the fermentation medium used is the widely used Pichia pastoris inorganic salt medium (glucose 30g / L, 85% phosphate 13ml / L, calcium sulfate dihydrate 0.58g / L, potassium sulfate 9.1g / L, magnesium sulfate heptahydrate 7.5g / L, potassium hydroxide 2g / L, PTM1 trace element 4ml / L).

[0040] Results: After fermentation for 40 hours, the conversion rate of canrone was 67.6%, and the first-pass yield of the purified 11α-hydroxycanrone was 54.3%. Compared with Example 1, both the conversion rate and the first-pass yield decreased by about 25%.

[0041] It can be seen that the fermentation medium formulation of the present invention is significantly superior to the traditional Pichia pastoris inorganic salt medium in promoting the expression of enzyme activity in the strain.

Claims

1. A process for the efficient conversion of canrenone to produce 11 alpha-hydroxy canrenone, characterized in that Having the following steps: ① Seed culture: Recombinant Pichia pastoris was inoculated into a shake flask containing seed culture medium and cultured until the OD of the bacterial culture reached a certain level. 600 When the value reaches 8-10, seed solution is obtained; ② Fermenter inoculation: the seed liquid obtained in step 1 is inoculated into a fermenter containing fermentation medium at an inoculation amount of 3-8%, and fermentation culture is carried out; the fermentation medium contains inositol; ③ Induction expression and substrate addition: the dissolved oxygen in the fermenter first decreases and then rises, and after the dissolved oxygen rises to more than 60%, methanol is added for induction expression; after 1.5-3h of induction expression, kanamycin is added to the fermenter until the conversion rate of kanamycin reaches more than 90%, the fermentation is ended, the fermentation broth is collected for subsequent extraction and purification, and 11α-hydroxykanamycin is obtained.

2. The process for the efficient conversion of canrenone to produce 11 a-hydroxy canrenone as claimed in claim 1 wherein: In the above step 1, the seed culture medium is YPD medium, and its formula is as follows: yeast extract 10g / L, peptone 20g / L, glucose 20g / L.

3. The process for the efficient conversion of kanamycin production of 11 a-hydroxy kanamycin according to claim 1, characterized by: In the above step 1, the seed culture temperature is 28-32℃, the rotation speed is 200-250rpm, and the time is 20-24h.

4. The process for the efficient conversion of canrenone to produce 11 a-hydroxy canrenone as claimed in claim 1 wherein: In the above step 2, the components of the fermentation medium are: glucose 25-35g / L, corn syrup dry powder 15-25g / L, potassium dihydrogen phosphate 10-15g / L, potassium phosphate dibasic trihydrate 1.0-2.0g / L, ammonium sulfate 1.5-2.5g / L, magnesium sulfate 0.3-0.7g / L, and inositol 0.01-0.05g / L.

5. The process for the efficient conversion of kanamycin production of 11 a-hydroxy kanamycin according to claim 1, characterized by: In the above step 2, the initial conditions of the fermentation culture are: temperature 28-32℃, pH 5.0-5.5, rotation speed 500-1000rpm, ventilation amount 1-2L / min, and dissolved oxygen controlled at more than 20%.

6. The process for the efficient conversion of kanamycin production of 11 a-hydroxy kanamycin according to claim 1, characterized by: In the above step 3, the initial flow rate of methanol is 1-2mL / h·L, and the highest flow rate of methanol is 5-6ml / h·L.

7. The process for the efficient conversion of kanamycin production of 11 alpha- hydroxykanamycin according to one of claims 1 to 6, characterized in that: In the above step 3, the feeding concentration of kanamycin is 20-40g / L.