Biosynthesis method and application of macrocycloalkanone compound based on cell engineering technology

Abstract

The invention belongs to the field of biosynthesis of macrocycloalkanone compounds, and particularly relates to a biosynthesis method and application of macrocycloalkanone compounds based on a cell engineering technology. The method comprises the following steps: culturing gland cells in a culture medium, adding reaction substrates aldehydes and fatty acid, and synthesizing the macrocycloalkanone compound from the aldehydes and fatty acid through cyclization reaction in the cells. The method provided by the invention overcomes the defects of complex process, low yield and high production control difficulty in artificial synthesis, and extremely low yield and unstable product properties in natural product extraction, and has the beneficial effects of realizing large-scale and engineering production, reducing the production cost and improving the yield and quality.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biosynthesis of macrocyclic ketones, in particular to a biosynthesis method of macrocyclic ketones based on cell engineering technology and application. BACKGROUND

[0002] Macrocyclic ketones are important compounds in industrial production and drug preparation, including dodecanedioic acid, dodecanediamine, nylon 12, nylon 612, nylon 1012, nylon 1212, nylon 12T, 3-methylcyclopentadecanone, muscone, cyclopentadecanone lactone, 5-cyclohexadecenone, cyclopentadecanone, 12-methyl-14-ketobicyclo[9,3,1]pentadecane, etc. Among them, nylon 12, 612, 1012, etc. are important raw materials for industrialized fiber manufacturing, and muscone, cyclopentadecanone, etc. are important drug raw materials.

[0003] However, at present, the main production methods of the above-mentioned macrocyclic ketones are mainly divided into two types: natural extraction and artificial synthesis. Taking muscone as an example, the main active ingredient of musk is L-muscone (R-Muscone), which has a bidirectional effect on the central nervous system, with small doses of excitement and large doses of inhibition, enhancing the hypoxic tolerance of the central nervous system and improving cerebral circulation; musk has obvious cardiotonic effect, can excite the heart, increase the amplitude of cardiac contraction, and enhance myocardial function; musk has preventive and therapeutic effects on ischemic heart disorders caused by thrombus; musk has certain anti-inflammatory effect, which is similar to that of hydrocortisone; musk has obvious excitatory and uterine contraction enhancing effect on the uterus, especially on the pregnant uterus, and the effect on the non-pregnant uterus occurs slowly but lasts long, muscone can significantly increase the frequency and intensity of uterine contractions, and has anti-implantation and anti-early pregnancy effects, and the anti-pregnancy effect becomes more significant as the gestational period prolongs; it has inhibitory effect on human tumor cells, and the effect is stronger at high concentration, and has killing effect on mouse ascitic tumor cells and sarcoma S180 cells.

[0004] The main production methods of muscone are mainly divided into natural extraction and artificial synthesis. Among them, the natural extraction method is to obtain musk from the scent sac of forest musk deer or other musk deer animals, and to form dry musk shape that can be used as medicine through air drying and other treatments. However, although artificial breeding is used, the population is still extremely rare, the time to go to the wild is short, the yield of single musk is low, and the quality of musk from different individuals is uneven, resulting in low yield of natural musk, and thus high production cost and raw material price, which is difficult to form large-scale application. Artificial synthesis of musk is mainly aimed at artificial synthesis of muscone. Artificially synthesized muscone is mainly in the form of racemate (L-muscone and D-muscone each accounts for about 50%), but the biological activity of muscone is mainly in the form of L-muscone, and the biological activity and medicinal properties of racemate are greatly affected. At present, there is no report on the biosynthesis method of musk and its active substances such as L-muscone.

[0005] In summary, the current production of macrocyclic ketone compounds, especially muscone, has obvious shortcomings whether by natural extraction or artificial synthesis. Through artificial cultivation, the product is little, the cycle is long, and the cost is high; chemical synthesis has the disadvantages of low yield, complex operation, complex route, high synthesis environment requirement, long cycle, etc. SUMMARY

[0006] In view of the above shortcomings in the prior art, the present application provides a new method for biosynthesis of macrocyclic ketone compounds based on cell engineering technology, which promotes the biosynthesis of macrocyclic ketone compounds, including muscone, effectively realizes the scaling and engineering of production, reduces the production cost, and improves the yield and quality.

[0007] In order to achieve the above-mentioned purpose of the present application, the present application provides a biosynthesis method of macrocyclic ketone compounds based on cell engineering technology:

[0008] The gland cells are cultured in the culture medium, and the reaction substrates aldehydes and fatty acids are added. In the cells, aldehydes and fatty acids are synthesized into macrocyclic ketone compounds through cyclization reaction, and the culture product is collected.

[0009] Or the gland cells, cell clusters or gland-like organs with androgen secretion function differentiated from adult stem cells or pluripotent stem cells, or mature gland cells, cell clusters or gland-like organs, are cultured, and the reaction substrates aldehydes and fatty acids are added. In the cells, aldehydes and fatty acids are synthesized into macrocyclic ketone compounds through cyclization reaction, and the culture product is collected.

[0010] In some embodiments, the culture of the gland cells in the culture medium is the expansion, induction and / or differentiation culture of the gland cells in the culture medium.

[0011] In some embodiments, the biosynthesis method comprises: inducing culture of the gland cells in the culture medium to induce differentiation of the gland cells to form gland cells, cell clusters, spheroids, three-dimensional cell colonies, or organoids with secretion function. The culture medium is a differentiation culture medium. The secretion function is androgen secretion function.

[0012] In some embodiments, the biosynthesis method comprises: culturing the gland cells in the culture medium by 2D or 3D culture method. The culture medium is an expansion culture medium.

[0013] The 3D culture of the present application, also referred to as 3D cell culture, is an in vitro culture technique that allows cells to grow and develop in three-dimensional space, forming cell aggregates or tissue-like structures with specific structures and functions. Compared with traditional two-dimensional (2D) cell culture, 3D culture can better simulate the natural environment of cells in vivo, including cell-cell interactions, extracellular matrix support, and responses to endogenous and exogenous stimuli. In 3D culture, cells not only grow in a plane but can form tissues with complex structures in three-dimensional space, such as spheroids, organoids, cell clusters, and three-dimensional cell colonies.

[0014] In more preferred embodiments, the biosynthesis method comprises: obtaining 2D or 3D cultured gland cells by culturing gland cells in a 2D or 3D culture method, and inducing the gland cells to differentiate in a differentiation medium to form cells, cell clusters, spheroids, three-dimensional cell colonies, or organoids with androgen secretion and macrocyclic ketone compound biosynthesis functions. Preferably, in the 2D or 3D culture, an expansion medium is used for culture.

[0015] The "3D cultured gland cells" obtained by 3D culture of the gland cells of the present application include a collection of cell types grown into cell clusters, spheroids, three-dimensional cell colonies, or organoids, and preferably, the 3D cultured gland cells include 3D gland cell clusters, spheroids, three-dimensional cell colonies, or organoids. More preferably, the 3D cultured gland cells are selected from 3D gland organoids. In the present application, cell clusters, spheroids, and three-dimensional cell colonies refer to the same or similar content, all of which represent 3D cultured gland cells, and three-dimensional cultures obtained by simulating in vivo growth.

[0016] In some embodiments, the biosynthesis method selectively includes or does not include expansion culture.

[0017] In some embodiments, the biosynthesis method includes simultaneously, sequentially, or separately performing expansion, induction, and / or differentiation culture.

[0018] In some embodiments, the gland cells are derived from the glands of musk deer (Moschus spp.) or muskrat (Ondatra zibethicus) animals, or from stem cells with multi-differentiation potential.

[0019] In more preferred embodiments, the gland cells are derived from gland of Moschus berezovskii, Moschus chrysogaster, Moschus moschiferus, Moschus leucogaster, Moschus fuscus, Ondatrazibethicus animal, or from stem cells with multi-differentiation potential.

[0020] In more preferred embodiments, the gland cells are derived from stem cells with multi-differentiation potential, and will be induced to differentiate to form gland cells, gland cell clusters or gland organoids with androgen secretion and macrocyclic ketone compound biosynthesis function through 2D or 3D culture.

[0021] In more preferred embodiments, the stem cells with multi-differentiation potential are selected from: induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

[0022] In more preferred embodiments, the adult stem cells or pluripotent stem cells are selected from: induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), mesenchymal stem cells (MSCs).

[0023] In more preferred embodiments, the glands are selected from: scent glands. Scent glands are glands around the genitalia that secrete androgens and macrocyclic ketone compounds such as muscone.

[0024] In more preferred embodiments, the 2D cultured gland cells are 2D adherent cultured gland cells.

[0025] In more preferred embodiments, the method further comprises one or more steps of isolating, pretreating or expanding culturing the gland tissue before culturing in the differentiation medium.

[0026] In more preferred embodiments, the method further comprises a step of differentiating the induced gland cells, gland cell clusters or gland organoids to form mature gland cells, gland cell clusters or gland organoids after culturing in the differentiation medium.

[0027] In more preferred embodiments, the method further comprises that the reaction substrate for synthesizing macrocyclic ketone compounds is before or at the same time or after the formation of mature gland cells, gland cell clusters or gland organoids.

[0028] In more preferred embodiments, the reaction substrate of the synthetic macrocyclic ketone compound is selected from the group consisting of: aldehydes and fatty acids; the aldehydes are selected from the group consisting of one or more of citronellal, citral, glutaraldehyde, and ombrofol; and the fatty acids are selected from the group consisting of one or more of linoleic acid, linolenic acid, arachidonic acid, palmitic acid, and docosadienoic acid. Preferably, the reaction substrate of the synthetic macrocyclic ketone compound is selected from the group consisting of: citral and arachidonic acid, or citronellal and palmitic acid, or glutaraldehyde and linolenic acid, or ombrofol and docosadienoic acid. When the reaction substrate of the synthetic macrocyclic ketone compound is selected from the group consisting of citral and arachidonic acid, the culture product is muscone; and when the reaction substrate of the synthetic macrocyclic ketone compound is selected from the group consisting of citronellal and palmitic acid, the culture product is cyclopentadecanone.

[0029] In some embodiments, the culture medium comprises: cytokines and small molecule components selected from the group consisting of one or more of hormone-like molecules or free fatty acids. The free fatty acid in the present application refers to the catabolic product of neutral fat, including the categories of cholesterol, triglyceride, phospholipid, etc. The free fatty acid added in the present application is a commercial product with comprehensive fatty acid composition.

[0030] In more preferred embodiments, the small molecule components further comprise one or more of BMP7 and DAPT. Preferably, the culture medium is a differentiation culture medium.

[0031] In more preferred embodiments, the cytokines are selected from the group consisting of one or more of Wnt agonists, EGF, and FGF.

[0032] In more preferred embodiments, the small molecule components are selected from the group consisting of hormone-like molecules and free fatty acids, and the hormone-like molecules are selected from the group consisting of one or more of androgens, prostaglandins, and estrogens. The differentiation culture medium provided by the present application (particularly containing androgens or / and free fatty acids) has a significant necessity for organoid differentiation and product synthesis. When only the androgen or free fatty acid component is removed, and other culture medium components and contents remain unchanged, the technical effect of significantly enhancing and promoting organoid differentiation and product synthesis cannot be achieved by the differentiation culture medium containing androgens or / and free fatty acids. Whether androgens and / or free fatty acids are added alone or in combination, the significant technical effect of enhancing organoid differentiation and increasing product synthesis can be achieved. In particular, the combination of androgens and free fatty acids has a more obvious synergistic effect of enhancing organoid differentiation and increasing product synthesis. Without the addition of androgens and free fatty acids, the differentiation culture medium has a relatively poor organoid growth and differentiation state and a very low product synthesis amount.

[0033] In more preferred embodiments, the culture medium further comprises: an antibiotic comprising a streptomycin solution, and / or a nutritional component selected from one or more of N2, B27.

[0034] In more preferred embodiments, the culture medium comprises: a hormone-like molecule and / or a free fatty acid, and further comprises one or more of a Wnt agonist, EGF, FGF, BMP7, DAPT, a streptomycin solution, N2, B27.

[0035] In more preferred embodiments, the Wnt agonist is selected from: a Wnt family member, Noggin, R-spondin, a BMP inhibitor, a GSK inhibitor, or an analogue thereof.

[0036] In still further preferred embodiments, the Wnt agonist is selected from: Wnt3A or an analogue thereof, Noggin, R-spondin, a BMP inhibitor, or a GSK inhibitor.

[0037] In more preferred embodiments, the culture medium comprises: EGF, FGF, an androgen, a prostaglandin, a free fatty acid, BMP7, DAPT, a streptomycin solution, N2, B27. Preferably, the prostaglandin is selected from: prostaglandin E2.

[0038] In more preferred embodiments, the androgen is selected from: dihydrotestosterone or testosterone undecanoate.

[0039] In more preferred embodiments, the culture product comprises: one or more of cyclopentadecenone, muscone, cyclododecene ketone, carbonyl bicyclo[9,3,1]pentadecane. Wherein, cyclopentadecenone is also known as cyclopentadecanone, pentacyclodecane ketone; muscone is also known as 3-methylcyclopentadecanone.

[0040] In some embodiments, the culture medium further comprises: a serum-free medium DMEM / F12 as a base medium.

[0041] The present application also provides the use of an adenohormone cell, or a 2D or 3D cultured adenohormone cell, or an adenohormone cell, adenohormone cell cluster or adenohormone organoid with androgen secretion function differentiated from an adult stem cell or a pluripotent stem cell, or an adenohormone cell, adenohormone cell cluster or adenohormone organoid matured by differentiation in the preparation or synthesis of a macrocyclic ketone compound.

[0042] In some embodiments, the adenohormone cell is derived from an adenohormone of a musk deer (Moschus spp.) or an ondatra zibethicus animal, or from a stem cell with multi-differentiation potential.

[0043] In more preferred embodiments, the gland cells are derived from glands of Moschus berezovskii, Moschus chrysogaster, Moschus moschiferus, Moschus leucogaster, Moschus fuscus, Ondatrazibethicus animals, or from stem cells with multi-differentiation potential.

[0044] In more preferred embodiments, the glands are selected from the group consisting of: scent glands. Scent glands are glands around the genitalia that secrete androstenone and other macrocyclic ketones.

[0045] In more preferred embodiments, the gland cells are derived from stem cells with multi-differentiation potential, and will be induced to differentiate in 2D or 3D culture to form gland cells, gland cell clusters or gland organoids with androstenone secretion and macrocyclic ketone biosynthesis functions.

[0046] In more preferred embodiments, the stem cells with multi-differentiation potential are selected from the group consisting of: induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

[0047] In more preferred embodiments, the adult stem cells or pluripotent stem cells are selected from the group consisting of: induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), mesenchymal stem cells (MSCs).

[0048] In some embodiments, the application further comprises: culturing the gland cells in a culture medium comprising: cytokines and small molecule components selected from one or more of: hormone-like molecules or free fatty acids.

[0049] In more preferred embodiments, the small molecule components further comprise: one or more of BMP7, DAPT. Preferably, the culture medium is a differentiation medium.

[0050] In more preferred embodiments, the cytokines are selected from one or more of: Wnt agonists, EGF, FGF.

[0051] In more preferred embodiments, the small molecule components are selected from: hormone-like molecules and free fatty acids, the hormone-like molecules are selected from one or more of: androgens, prostaglandins, estrogens.

[0052] In more preferred embodiments, the culture medium further comprises: antibiotics and / or nutritional components, the antibiotics comprise penicillin-streptomycin solution, the nutritional components are selected from one or more of: N2, B27.

[0053] In more preferred embodiments, the culture medium comprises: a hormone-like molecule and / or a free fatty acid, and further comprises one or more of a Wnt agonist, EGF, FGF, BMP7, DAPT, streptomycin solution, N2, B27.

[0054] In more preferred embodiments, the Wnt agonist is selected from the group consisting of: a Wnt family member, Noggin, R-spondin, a BMP inhibitor, a GSK inhibitor, or an analog thereof.

[0055] In still more preferred embodiments, the Wnt agonist is selected from the group consisting of: Wnt3A or an analog thereof, Noggin, R-spondin, a BMP inhibitor, or a GSK inhibitor.

[0056] In more preferred embodiments, the culture medium comprises: EGF, FGF, an androgen, a prostaglandin, a free fatty acid, BMP7, DAPT, streptomycin solution, N2, B27. Preferably, the prostaglandin is selected from the group consisting of: prostaglandin E2.

[0057] In more preferred embodiments, the androgen is selected from the group consisting of: dihydrotestosterone or testosterone undecanoate.

[0058] In some embodiments, the use further comprises culturing the glandular cells in the culture medium, adding a reaction substrate aldehyde and a fatty acid, and synthesizing the macrocyclic ketone compound in the cells via a cyclization reaction.

[0059] In more preferred embodiments, the macrocyclic ketone compound comprises one or more of: cyclopentadecanone, musk ketone, cyclohexadecenone, carbonylbicyclo[9,3,1]pentadecane.

[0060] In some embodiments, the culturing of the glandular cells in the culture medium is an expansion, induction, and / or differentiation culturing of the glandular cells.

[0061] In some embodiments, the use further comprises: inducing culturing of the glandular cells in the culture medium, inducing differentiation of the glandular cells to form glandular cells, cell clusters, spheroids, three-dimensional cell colonies, or organoids having a secretory function. The culture medium is a differentiation culture medium. The secretory function is an androgen secretory function.

[0062] In some embodiments, the use further comprises: culturing the glandular cells in the culture medium using a 2D or 3D culturing method. The culture medium is an expansion culture medium.

[0063] In more preferred embodiments, the use further comprises: culturing the gland cells in 2D or 3D culture method to obtain 2D or 3D cultured gland cells, inducing culture of the 2D or 3D cultured gland cells in differentiation medium, inducing differentiation of the gland cells to form cells, cell clusters, spheroids, three-dimensional cell colonies, or organoids with androgen secretion and macrolactone biosynthesis function. Preferably, in the 2D or 3D culture, the culture is performed using expansion medium.

[0064] In some embodiments, the use selectively comprises or does not comprise expansion culture.

[0065] In some embodiments, the use comprises performing expansion, induction and / or differentiation culture simultaneously, sequentially or separately.

[0066] In more preferred embodiments, the 2D cultured gland cells are 2D adherent cultured gland cells.

[0067] In more preferred embodiments, the use further comprises one or more steps of separation, pretreatment or expansion culture of the gland tissue before culturing in differentiation medium.

[0068] In more preferred embodiments, the use further comprises a step of inducing differentiation of the induced gland cells, gland cell clusters or gland organoids to form mature gland cells, gland cell clusters or gland organoids after culturing in differentiation medium. In more preferred embodiments, the synthetic macrolactone reaction substrate is before or simultaneously or after the formation of mature gland cells.

[0069] In more preferred embodiments, the synthetic macrolactone reaction substrate is selected from: aldehydes and fatty acids; the aldehydes are selected from one or more of citronellal, citral, glutaraldehyde, ombrofol; the fatty acids are selected from one or more of linoleic acid, linolenic acid, arachidonic acid, palmitic acid, docosadienoic acid. Preferably, the synthetic macrolactone reaction substrate is selected from: citral and arachidonic acid, or citronellal and palmitic acid, or glutaraldehyde and linolenic acid, or ombrofol and docosadienoic acid. When the synthetic macrolactone reaction substrate is selected from: citral and arachidonic acid, the culture product is musk ketone; when the synthetic macrolactone reaction substrate is selected from: citronellal and palmitic acid, the culture product is cyclopentadecanone.

[0070] In some embodiments, the method comprises one or more steps of washing, tissue separation and mincing, digestion, filtration.

[0071] In more preferred embodiments, the tissue separation and mincing is, after the tissue is removed from the body, the skin and muscle tissue is peeled off to obtain the subcutaneous glandular tissue, and the tissue is minced to about 1-2 mm using sterilized ophthalmic scissors. 3 .

[0072] In more preferred embodiments, the digestion is, the addition of the digestion solution, and the digestion of the glandular cells at 37°C.

[0073] In more preferred embodiments, the digestion is, the addition of the digestion solution, and the digestion of the glandular cells at 37°C.

[0074] In more preferred embodiments, the washing is, the repeated washing of the tissue sample with cold HBSS solution containing 2% of the secondary antibody.

[0075] In more preferred embodiments, the tissue sample is a 0.5 cm 3 -3 cm 3 tissue sample.

[0076] In some embodiments, the method further comprises, after the digestion is completed, the addition of a double volume of cold HBSS solution to dilute the digestion solution and the filtration operation.

[0077] In more preferred embodiments, the filtration operation is, the filtration of the collected filtrate using a 100 μm cell screen, the centrifugation at 1200 rpm for 3 minutes, and the collection of the cell precipitate after the supernatant is removed.

[0078] The present application also provides a differentiation medium, comprising: a cytokine and a small molecule component, the small molecule component being selected from one or more of: a hormone-like molecule or a free fatty acid.

[0079] In some embodiments, the small molecule component further comprises: one or more of BMP7, DAPT.

[0080] In some embodiments, the cytokine is selected from one or more of: a Wnt agonist, EGF, FGF.

[0081] In some embodiments, the small molecule component is selected from: a hormone-like molecule and a free fatty acid, the hormone-like molecule being selected from one or more of: an androgen, a prostaglandin, an estrogen.

[0082] In more preferred embodiments, the androgen is selected from: dihydrotestosterone or testosterone undecanoate.

[0083] In a more preferred embodiment, the culture medium further comprises: antibiotics and / or nutrients, the antibiotics include penicillin-streptomycin solution, and the nutrients are selected from: one or more of N2 and B27.

[0084] In a more preferred embodiment, the culture medium includes: hormone molecules and / or free fatty acids, and also includes one or more of Wnt agonists, EGF, FGF, BMP7, DAPT, penicillin-streptomycin solution N2, and B27.

[0085] In a more preferred embodiment, the Wnt agonist is selected from the group consisting of: a Wnt family member, Noggin, R-spondin, a BMP inhibitor, a GSK inhibitor, or an analog thereof.

[0086] In a further preferred embodiment, the Wnt agonist is selected from the group consisting of: Wnt3A or an analog thereof, Noggin, R-spondin, a BMP inhibitor or a GSK inhibitor.

[0087] In a more preferred embodiment, the culture medium comprises: EGF, FGF, androgen, prostaglandin, free fatty acid, BMP7, DAPT. Preferably, the prostaglandin is selected from: prostaglandin E2.

[0088] In a more preferred embodiment, the culture medium comprises: Wnt3A or Noggin or R-spondin or BMP inhibitor or GSK inhibitor, EGF, FGF, androgen, prostaglandin, free fatty acid, BMP7, DAPT. Preferably, the prostaglandin is selected from: prostaglandin E2.

[0089] In a more preferred embodiment, the culture medium comprises: Wnt3A, Noggin, EGF, FGF, androgen, prostaglandin, free fatty acid, BMP7, DAPT. Preferably, the prostaglandin is selected from: prostaglandin E2.

[0090] In a more preferred embodiment, the culture medium comprises: EGF, FGF, androgen, prostaglandin, free fatty acid, BMP7, DAPT, penicillin-streptomycin solution, N2, B27. Preferably, the prostaglandin is selected from: prostaglandin E2.

[0091] In more preferred embodiments, the culture medium comprises: 5-50 ng / ml EGF, 5-100 ng / ml FGF, 10-250 ng / ml androgen, 50-1000 ng / ml prostaglandin, 1-5X free fatty acid, 1-100 ng / ml BMP7, 10-500 nM DAPT, 1-5X streptomycin solution, 1-10X N2, 1-10X B27. Preferably, the prostaglandin is selected from the group consisting of prostaglandin E2.

[0092] In more preferred embodiments, the culture medium comprises: 25 ng / ml EGF, 50 ng / ml FGF, 200 ng / ml androgen, 500 ng / ml prostaglandin, 2X free fatty acid, 10 ng / ml BMP7, 50 nM DAPT, 1-5X streptomycin solution, 1-10X N2, 1-10X B27. Preferably, the prostaglandin is selected from the group consisting of prostaglandin E2.

[0093] In more preferred embodiments, the culture medium comprises: 10 ng / ml Wnt3A, 10 ng / ml Noggin, 25 ng / ml EGF, 50 ng / ml FGF, 200 ng / ml androgen, 500 ng / ml prostaglandin, 1-5X free fatty acid, 10 ng / ml BMP7, 50 nM DAPT, 1-5X streptomycin solution, 1-10X N2, 1-10X B27. Preferably, the prostaglandin is selected from the group consisting of prostaglandin E2.

[0094] In more preferred embodiments, the androgen is selected from the group consisting of dihydrotestosterone or testosterone undecanoate.

[0095] The present application also provides the use of the above-mentioned differentiation medium in differentiating glandular cells, glandular cell clusters or glandular organoids or in preparing or synthesizing macrocyclic ketone compounds.

[0096] The present application also provides a method for 2D and 3D cell culture of scent gland tissue, comprising: performing 2D and 3D cell culture on glandular cells extracted after digestion through an expansion medium, wherein the expansion medium comprises one or more of Wnt agonist, R-spondin, Noggin, EGF, FGF, androgen, prostaglandin, free fatty acid, streptomycin solution, N2, and B27. Preferably, the prostaglandin is selected from the group consisting of prostaglandin E2.

[0097] In some embodiments, the expansion medium comprises: 5-1000 ng / ml Wnt3A, 10-5000 ng / ml R-spondin, 10-1000 ng / ml Noggin, 5-100 ng / ml EGF, 5-100 ng / ml FGF, 0.1-100 ng / ml androgen, 10-1000 ng / ml prostaglandin E2, 1-10X free fatty acid, 1-10X streptomycin solution, 1-10X N2, 1-10X B27.

[0098] In more preferred embodiments, the expansion medium comprises: 10-100 ng / ml Wnt3A, 50-2000 ng / ml R-spondin, 10-500 ng / ml Noggin, 10-100 ng / ml EGF, 10-100 ng / ml FGF, 0.1-50 ng / ml androgen, 10-500 ng / ml prostaglandin E2, 1-5X free fatty acid, 1-5X streptomycin solution, 1-10X N2, 1-10X B27.

[0099] In more preferred embodiments, the androgen is selected from the group consisting of: dihydrotestosterone or testosterone undecanoate.

[0100] In more preferred embodiments, the expansion medium comprises: 100 ng / ml Wnt3A, 500 ng / ml R-spondin, 100 ng / ml Noggin, 50 ng / ml EGF, 20 ng / ml FGF, 10 ng / ml dihydrotestosterone, 100 ng / ml prostaglandin E2, 1X free fatty acid, 1X streptomycin solution, 1X N2, 1X B27.

[0101] In more preferred embodiments, the expansion medium comprises: 50 ng / ml Wnt3A, 1000 ng / ml R-spondin, 200 ng / ml Noggin, 20 ng / ml EGF, 10 ng / ml FGF, 30 ng / ml testosterone undecanoate, 100 ng / ml prostaglandin E2, 2X free fatty acid, 1X streptomycin solution, 2X N2, 2X B27.

[0102] In more preferred embodiments, the expansion medium comprises: 30 ng / ml Wnt3A, 100 ng / ml R-spondin, 50 ng / ml Noggin, 50 ng / ml EGF, 50 ng / ml FGF, 60 ng / ml dihydrotestosterone, 300 ng / ml prostaglandin E2, 1X free fatty acid, 1X streptomycin solution, 2X N2, 2X B27.

[0103] In more preferred embodiments, the expansion medium further comprises serum-free medium DMEM / F12.

[0104] After the extracted glandular cells are subjected to 2D and 3D cell culture by the expansion medium in the culture method of the present application, the glandular cells subjected to 2D and 3D culture can be obtained, which have the ability of sustained expansion, subculture, and can be frozen and recovered. Preferably, the glandular cells are scent gland cells.

[0105] In some embodiments, the method comprises that the culture is gel culture or suspension culture.

[0106] In more preferred embodiments, the gel culture comprises that matrigel is mixed with the cell suspension uniformly on ice at a ratio of 1:2, inoculated in a 6-well plate, placed in a 37°C, 5% CO2 incubator for 30 minutes, and then the culture medium of the present application is added after the matrigel solidifies.

[0107] In more preferred embodiments, the method further comprises that red blood cells are lysed and / or cell counting before the gel culture.

[0108] In more preferred embodiments, the lysis of red blood cells comprises that 1-3 ml of red blood cell lysis solution is added to resuspend the precipitate, left to stand at room temperature for 3 minutes, and then 3-6 ml of DMEM / F12 is added to terminate the lysis, centrifuged at 1200 rpm for 3 minutes, and the supernatant is removed to collect the cell precipitate.

[0109] In more preferred embodiments, if there are not many red blood cells, the step of lysing red blood cells can be omitted.

[0110] In more preferred embodiments, the cell counting comprises that 3-5 ml of DMEM / F12 is used to resuspend the cell precipitate, and an appropriate amount of the suspension is taken for cell counting, and the cell inoculation culture density is adjusted according to the counting result.

[0111] In more preferred embodiments, the gel culture comprises that matrigel is mixed with the cell suspension uniformly on ice at a ratio of 1:2, inoculated in a 6-well plate, placed in a 37°C, 5% CO2 incubator for 30 minutes, and then the culture medium of the present application is added after the matrigel solidifies.

[0112] In more preferred embodiments, the gel culture further comprises that the liquid medium is replaced every 2-3 days, and the glandular organ is harvested after 4-7 days of culture.

[0113] In more preferred embodiments, the suspension culture comprises that the cell suspension is inoculated into a culture plate or dish coated with ultralow adhesion material.

[0114] In more preferred embodiments, the suspension culture further comprises placing the culture plate or dish in a horizontal shaker or bioreactor, adjusting the shaker or bioreactor speed to 100-150 rpm, and adjusting the culture temperature to 26-37 °C.

[0115] In more preferred embodiments, the suspension culture further comprises placing the culture plate or dish in a horizontal shaker or bioreactor, adjusting the shaker or bioreactor speed to 100-150 rpm, and adjusting the culture temperature to 26-37 °C.

[0116] In more preferred embodiments, the suspension culture further comprises centrifuging the cells at 1500 rpm for 3-5 min, discarding the supernatant, and resuspending the cells in the medium described herein.

[0117] The present application also provides an expansion medium comprising one or more of a Wnt agonist, R-spondin, Noggin, EGF, FGF, androgen, prostaglandin E2, free fatty acid, streptomycin solution, N2, and B27.

[0118] In some embodiments, the expansion medium comprises 5-1000 ng / ml Wnt3A, 10-5000 ng / ml R-spondin, 10-1000 ng / ml Noggin, 5-100 ng / ml EGF, 5-100 ng / ml FGF, 0.1-100 ng / ml androgen, 10-1000 ng / ml prostaglandin E2, 1-10X free fatty acid, 1-10X streptomycin solution, 1-10X N2, and 1-10X B27.

[0119] In more preferred embodiments, the expansion medium comprises 10-100 ng / ml Wnt3A, 50-2000 ng / ml R-spondin, 10-500 ng / ml Noggin, 10-100 ng / ml EGF, 10-100 ng / ml FGF, 0.1-50 ng / ml androgen, 10-500 ng / ml prostaglandin E2, 1-5X free fatty acid, 1-5X streptomycin solution, 1-10X N2, and 1-10X B27.

[0120] In more preferred embodiments, the androgen is selected from the group consisting of dihydrotestosterone or testosterone undecanoate.

[0121] In a more preferred embodiment, the expansion medium comprises: 100 ng / ml Wnt3A, 500 ng / ml R-spondin, 100 ng / ml Noggin, 50 ng / ml EGF, 20 ng / ml FGF, 10 ng / ml dihydrotestosterone, 100 ng / ml prostaglandin E2, 1X free fatty acid, 1X streptomycin solution, 1X N2, 1X B27.

[0122] In a more preferred embodiment, the expansion medium comprises: 50 ng / ml Wnt3A, 1000 ng / ml R-spondin, 200 ng / ml Noggin, 20 ng / ml EGF, 10 ng / ml FGF, 30 ng / ml testosterone undecanoate, 100 ng / ml prostaglandin E2, 2X free fatty acid, 1X streptomycin solution, 2X N2, 2X B27.

[0123] In a more preferred embodiment, the expansion medium comprises: 30 ng / ml Wnt3A, 100 ng / ml R-spondin, 50 ng / ml Noggin, 50 ng / ml EGF, 50 ng / ml FGF, 60 ng / ml dihydrotestosterone, 300 ng / ml prostaglandin E2, 2X free fatty acid, 1X streptomycin solution, 2X N2, 2X B27.

[0124] In a more preferred embodiment, the expansion medium further comprises a serum-free medium DMEM / F12.

[0125] The application also provides the use of the above-mentioned expansion medium for expanding glandular cells, glandular cell clusters or glandular organoids or for preparing or synthesizing macrocyclic ketone compounds.

[0126] The application also provides a glandular organoid prepared or obtained by the above-mentioned method, and the use of the glandular organoid prepared or obtained by the above-mentioned method for preparing or synthesizing macrocyclic ketone compounds.

[0127] The application has the following beneficial effects:

[0128] (1) The application initiatively proposes a method and application for biosynthesizing macrocyclic ketone compounds based on cell engineering, i.e., a technical method for cell engineering culture and induction biosynthesis using mammalian cells.

[0129] (2) The application provides an innovative biosynthesis method of musk key active substances, which realizes biosynthesis of muscone by using in-vitro 2D and 3D organoids of scent sac cells, and can realize large-scale and engineering production of muscone, reduce production cost, and improve yield and quality. In the production of musk, the existing musk yield can be significantly improved, and the application of musk in more drug research is promoted.

[0130] (3) The application provides separation, digestion, in-vitro primary culture, construction of amplifiable and preserved scent sac engineering cells of scent gland cells, which can continuously and efficiently produce muscone and other active substances in-vitro, and provide strong guarantee for large-scale, engineering production of products, yield and quality of products.

[0131] (4) The amplification medium provided by the application can effectively form gland organoids from gland cells by self-assembly, and the currently marketed medium cannot effectively form gland organoids from gland cells by self-assembly.

[0132] (5) The differentiation medium provided by the application (especially containing androgen or / and free fatty acid) has remarkable necessity for organoid differentiation and product synthesis. When only the component of androgen or free fatty acid is removed, and other components and contents of the medium remain unchanged, the technical effect of the differentiation medium containing androgen or / and free fatty acid on the remarkable enhancement and promotion of organoid differentiation and product synthesis cannot be achieved. Whether androgen and / or free fatty acid is added alone or in combination, the remarkable technical effect of enhancing organoid differentiation and increasing product synthesis can be achieved. In particular, the combination of androgen and free fatty acid has more obvious synergistic effect of enhancing organoid differentiation and increasing product synthesis. The organoid growth and differentiation state of the differentiation medium without androgen and free fatty acid is relatively poor, and the product synthesis amount is extremely low. BRIEF DESCRIPTION OF DRAWINGS

[0133] Figure 1 The figure shows the gland organoids obtained by culturing in Example 3;

[0134] Figure 2 The figure shows the gland organoids obtained by culturing in Example 4;

[0135] Figure 3 The figure shows the gland organoids obtained by culturing in Example 5;

[0136] Figure 4 The figure shows the gas chromatography-mass spectrometry analysis result of muscone obtained by biosynthesis of the application;

[0137] Figure 5 The figure shows the gas chromatography-mass spectrometry analysis result of cyclopentadecanone obtained by biosynthesis of the application;

[0138] Figure 6 The results of the comparison of the culture effects of the amplification medium of the present application and other market media are shown;

[0139] Figure 7 The results of the comparison of the product synthesis effects before and after removing the androgen and the androgen and free fatty acid in the differentiation medium of the present application are shown;

[0140] Figure 8 The results of the comparison of the product synthesis effects before and after removing the free fatty acid in the differentiation medium of the present application are shown. DETAILED DESCRIPTION

[0141] In order to make the purpose, technical scheme and advantages of the present application more clear, the following further describes the compounds, compositions and applications of the present application through examples. It should be understood that the specific examples described herein are only used to explain the present application, and are not used to limit the present application.

[0142] Example 1 Isolation, digestion and cell extraction of musk deer gland

[0143] 1. Washing: Place the musk deer gland tissue sample (about 0.5cm 3 -3cm 3 ) in a 50ml centrifuge tube, and repeatedly wash the tissue with cold HBSS solution containing 2% Dextran for 3-5 times.

[0144] 2. Digestion: Cut the sample into small pieces as much as possible with a sterile ophthalmic scissors, and add 8-10ml of gland trypsin digestion solution (Gibco) to shake and digest at 37°C. Observe the tissue digestion under a microscope every 5 minutes, and use a pipette to gently blow the tissue during the observation. After about 20-30 minutes, the tissue is digested into small cell clumps with good activity.

[0145] 3. Filtration: After digestion, dilute the sample with double volume of cold HBSS solution, and perform filtration operation. Use a 100μm cell screen to collect the filtrate, centrifuge at 1200rpm for 3 minutes, and collect the cell precipitate after removing the supernatant.

[0146] Example 2 Isolation, digestion and cell extraction of musk deer gland

[0147] 1. Washing: Place the musk deer gland tissue sample (about 0.5cm 3 -3cm 3 ) in a 50ml centrifuge tube, and repeatedly wash the tissue with cold HBSS solution containing 2% Dextran for 3-5 times.

[0148] 2. Digestion: Use sterile ophthalmic scissors to mince the specimen as finely as possible. Add 8-10 ml of glandular digestion solution and shake in a 37°C shaker. Observe the tissue digestion progress under a microscope every 5 minutes, gently pipetting the tissue during digestion. Digest for approximately 20-30 minutes, until the tissue is reduced to smaller, more active cell clumps.

[0149] 3. Filtration: After digestion is complete, dilute the solution with twice the volume of cold HBSS and filter. Collect the filtrate using a 100 μm cell sieve and centrifuge at 1200 rpm for 3 minutes. Remove the supernatant and collect the cell pellet.

[0150] Example 3 3D organoid culture of musk deer gland tissue (gel drop method or gel culture method)

[0151] 1. Lyse red blood cells: Resuspend the pellet in 1-3 ml of red blood cell lysis buffer. Let stand at room temperature for 3 minutes. Stop lysis with 3-6 ml of DMEM / F12. Centrifuge at 1200 rpm for 3 minutes and remove the supernatant to collect the cell pellet. This step can be omitted if there are not many red blood cells.

[0152] 2. Cell Count: Resuspend the cell pellet in 3-5 ml of DMEM / F12 and take an appropriate amount of suspension for cell counting. Adjust the cell seeding density based on the count results.

[0153] 3. Inoculation and culture: Mix matrigel and cell suspension at a ratio of 1:2 on ice. Inoculate into 6-well plates and place in a 37°C, 5% CO2 incubator for 30 minutes. After the matrigel gel solidifies, add expansion medium composed of:

[0154] Serum-free culture medium DMEM / F12, and including Wnt3A (100ng / ml), R-spondin (500ng / ml), Noggin (100ng / ml), EGF (50ng / ml), FGF (20ng / ml), dihydrotestosterone (10ng / ml), prostaglandin E2 (100ng / ml), free fatty acids (1X), penicillin-streptomycin solution (1X), N2 (1X), B27 (1X).

[0155] 4. Replace the liquid culture medium every 2-3 days. Glandular organoids can be obtained after 4-7 days of culture. The cultured glandular organoids are as follows: Figure 1 shown.

[0156] Example 4 3D cell culture of musk gland tissue (suspension method)

[0157] 1. Lysis of red blood cells: Add 1-3 ml of red blood cell lysis solution to resuspend the pellet, stand at room temperature for 3 minutes, terminate lysis with 3-6 ml of DMEM / F12, centrifuge at 1200 rpm for 3 minutes, remove the supernatant and collect the cell pellet. If there are not many red blood cells, this step can be omitted.

[0158] 2. Cell counting: Resuspend the cell pellet with 3-5 ml of DMEM / F12, take an appropriate amount of the suspension for cell counting. Adjust the cell seeding culture density according to the counting results.

[0159] 3. Seeding culture: Seed the cell suspension into an ultra-low adhesion 6-well culture plate, supplement the medium to 3 ml per well, and place the culture plate in a horizontal shaker, 120 rpm, 37°C, 5% CO2 culture.

[0160] Serum-free medium DMEM / F12, and including Wnt3A (50 ng / ml), R-spondin (1000 ng / ml), Noggin (200 ng / ml), EGF (20 ng / ml), FGF (10 ng / ml), androgen (testosterone undecanoate, 30 ng / ml), prostaglandin E2 (100 ng / ml), free fatty acid (2X), penicillin-streptomycin solution (1X), N2 (2X), B27 (2X).

[0161] 4. Replace the liquid medium every 2-3 days, and after 4-7 days of culture, the glandular organoids can be obtained, and the glandular organoids obtained are as shown in Figure 2

[0162] Example 5 3D cell culture of musk deer gland tissue

[0163] 1. Lysis of red blood cells: Add 1-3 ml of red blood cell lysis solution to resuspend the pellet, stand at room temperature for 3 minutes, terminate lysis with 3-6 ml of DMEM / F12, centrifuge at 1200 rpm for 3 minutes, remove the supernatant and collect the cell pellet. If there are not many red blood cells, this step can be omitted.

[0164] 2. Cell counting: Resuspend the cell pellet with 3-5 ml of DMEM / F12, take an appropriate amount of the suspension for cell counting. Adjust the cell seeding culture density according to the counting results.

[0165] 3. Seeding culture: Seed the cell suspension into an ultra-low adhesion 6-well culture plate, supplement the medium to 3 ml per well, and place the culture plate in a horizontal shaker, 120 rpm, 37°C, 5% CO2 culture.

[0166] ​Serum-free medium DMEM / F12, with cytokines, including EGF (25 ng / ml), FGF (50 ng / ml), Dihydrotestosterone (200 ng / ml), Prostaglandin E2 (500 ng / ml), Free Fatty Acid (2X), BMP7 (10 ng / ml), DAPT (50 nM), Penicillin-Streptomycin Solution (1X), N2 (1X), B27 (1X);

[0167] 4. Replace the liquid medium every 2-3 days with half the volume, and replace the liquid medium once every 2-3 days. After 4-7 days of culture, the glandular organ can be obtained, and the glandular organ obtained is shown in Figure 3 .

[0168] Example 6 Induced differentiation of musk gland cells and biosynthesis of muscone

[0169] 1. For the glandular organ cultured in Example 3, the expansion medium is aspirated and replaced with organoid differentiation medium, and the organoid differentiation medium comprises:

[0170] Serum-free medium DMEM / F12, with cytokines, including EGF (25 ng / ml), FGF (50 ng / ml), Dihydrotestosterone (200 ng / ml), Prostaglandin E2 (500 ng / ml), Free Fatty Acid (2X), BMP7 (10 ng / ml), DAPT (50 nM), Penicillin-Streptomycin Solution (1X), N2 (1X), B27 (1X);

[0171] 2. After replacing with the differentiation medium, replace the liquid every 3 days, and repeat 3 times;

[0172] 3. After 9 days of differentiation, add the reaction substrate: citral (500 nM), arachidonic acid (1000 nM) to the differentiation medium in step 1 to prepare a reaction medium;

[0173] 4. Replace the reaction medium every 5 days, and collect the medium;

[0174] 5. Freeze-dry the collected medium, separate the components by gel chromatography, extract the muscone component, and identify the muscone by gas chromatography-mass spectrometry. The identification result of muscone biosynthesized by the organoid is shown in Figure 4 .

[0175] Example 7 Induced differentiation of musk gland cells and biosynthesis of cyclopentadecenone

[0176] 1. For the musk gland cells cultured in Example 5, the expansion medium is aspirated and replaced with a differentiation medium, and the organoid differentiation medium comprises:

[0177] Serum-free medium DMEM / F12, and including Wnt3A (10 ng / ml), Noggin (10 ng / ml), EGF (25 ng / ml), FGF (50 ng / ml), dihydrotestosterone (200 ng / ml), prostaglandin E2 (500 ng / ml), free fatty acid (2X), BMP7 (10 ng / ml), DAPT (50 nM), penicillin-streptomycin solution (1X), N2 (1X), B27 (1X);

[0178] 2. After changing to the differentiation medium, replace the liquid every 3 days, and repeat 3 times;

[0179] 3. After 9 days of differentiation, add the reaction substrate: citronellal (1000 nM), palmitic acid (1000 nM) to the differentiation medium described in step 1 to prepare a reaction medium;

[0180] 4. Replace the liquid every 5 days using the reaction medium, and collect the medium;

[0181] 5. Freeze-dry the collected medium, separate the components by gel chromatography, extract the cyclopentadecanone component, and identify the cyclopentadecanone by gas chromatography-mass spectrometry, and the results are shown in Figure 5 .

[0182] Comparative Example 1. Comparison of culture effects of the medium described in the application and other market media

[0183] 1. Lysis of red blood cells: add 1-3 ml of red blood cell lysis solution to resuspend the precipitate, stand at room temperature for 3 minutes, terminate lysis with 3-6 ml of DMEM / F12, centrifuge at 1200 rpm for 3 minutes, remove the supernatant and collect the cell precipitate. If there are not many red blood cells, this step can be omitted.

[0184] 2. Cell counting: use 3-5 ml of DMEM / F12 to resuspend the cell precipitate, take an appropriate amount of suspension for cell counting. Adjust the cell inoculation culture density according to the counting results.

[0185] 3. Divide the cells into two groups: ① the medium group described in the application (use the medium in Example 3), and ② the market medium group (IntestiCult, STEMCELL)

[0186] 4. Inoculation culture: mix the matrigel and cell suspension according to 1:2 on ice. Inoculate in a 6-well plate, and place in a 37℃, 5% CO2 incubator for 30 minutes. After the matrigel solidifies, add the medium, wherein group ① adds the medium described in Example 3, and group ② adds the market medium:

[0187] 4. Replace the liquid culture medium every 2-3 days. Glandular organoids can be obtained after 4-7 days of culture. The cultured glandular organoids are as follows: Figure 6 As shown, the use of the culture medium of the present invention can effectively self-assemble glandular cells into glandular organoids, while commercially available culture medium cannot effectively self-assemble glandular cells into glandular organoids.

[0188] Comparative Example 2. Comparison of the differentiation and synthesis effects before and after the removal of androgens and the addition of androgens and free fatty acids in the differentiation medium of the present invention

[0189] 1. The glandular organoids cultured in Example 3 were divided into three groups: Group 1 used the differentiation medium of the present invention (using the medium of Example 6), Group 2 only removed the androgen, and Group 3 removed the androgen and free fatty acids, while other medium compositions and contents remained unchanged.

[0190] 2. Aspirate the expansion medium and replace it with organoid differentiation medium, as defined in step 1;

[0191] 3. After changing to differentiation medium, change the medium every 3 days, repeat 3 times;

[0192] 4. After 9 days of differentiation, the reaction substrates, citral (500 nM) and arachidonic acid (1000 nM), were added to the differentiation medium described in step 1 to prepare a reaction medium;

[0193] 5. Use reaction medium to change the medium every 5 days and collect the medium;

[0194] 6. The collected culture medium is freeze-dried and separated by gel chromatography to extract the components containing musk ketone. The musk ketone can be identified by gas chromatography-mass spectrometry. The identification results of musk ketone synthesized by organoid biosynthesis are as follows: Figure 7 As shown, the expression level (relative peak area, AUC) of musk ketone biosynthesized using the differentiation medium of the present invention is about 6, while the relative peak area value of musk ketone biosynthesized using the differentiation medium that does not contain androgens is 1.8, and the numerical difference is relatively obvious. After removing androgens and free fatty acids at the same time, the musk ketone content is less than 0.5, and the expression level is extremely low, which is significantly reduced compared to the expression levels of musk ketone in the two groups of adding androgens and free fatty acids (group 1) and adding free fatty acids (group 2). The results show that androgens and / or free fatty acids are significantly necessary for organoid differentiation and product synthesis. Moreover, the simultaneous addition of androgens and free fatty acids can enhance the differentiation effect of organoids and increase the amount of product synthesis, and has a synergistic effect.

[0195] Comparative Example 3. Comparison of differentiation and synthesis effects before and after removal of free fatty acids in the differentiation medium of the present invention

[0196] 1. The glandular organoids cultured in Example 3 were divided into two groups: ① the differentiation medium of the present invention (using the medium of Example 6), and group ② in which only the free fatty acids were removed, while the other medium compositions and contents remained unchanged.

[0197] 2. Aspirate the expansion medium and replace it with organoid differentiation medium, as defined in step 1;

[0198] 3. After changing to differentiation medium, change the medium every 3 days, repeat 3 times;

[0199] 4. After 9 days of differentiation, the reaction substrates, citronellal (500 nM) and palmitic acid (1000 nM), were added to the differentiation medium described in step 1 to prepare a reaction medium;

[0200] 5. Use reaction medium to change the medium every 5 days and collect the medium;

[0201] 6. The collected culture medium is freeze-dried and separated by gel chromatography to extract the cyclopentadecanone component. The cyclopentadecanone can be identified by gas chromatography-mass spectrometry. The identification results of cyclopentadecanone synthesized by organoid biosynthesis are as follows: Figure 8 As shown, the expression level (relative peak area, AUC) of cyclopentadecanone biosynthesized using the differentiation medium of the present invention is 8.22, while the expression level (relative peak area, AUC) of cyclopentadecanone biosynthesized using the differentiation medium that does not contain free fatty acids is 0.23, and the numerical difference is quite significant. The results show that free fatty acids are significantly necessary for organoid differentiation and product synthesis. Adding free fatty acids can significantly enhance the differentiation effect of organoids and increase the amount of product synthesis.

[0202] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for biosynthesis of macrocyclic ketone compounds based on cell engineering technology, characterized in that, culturing gland cells in a culture medium, adding reaction substrates aldehydes and fatty acids, synthesizing macrocyclic ketone compounds from aldehydes and fatty acids through cyclization reaction in the cells, and collecting the culture product; or culturing gland cells, gland cell clusters or gland organoids with androgen secretion differentiated from adult stem cells or pluripotent stem cells, or mature gland cells, gland cell clusters or gland organoids differentiated, adding reaction substrates aldehydes and fatty acids, synthesizing macrocyclic ketone compounds from aldehydes and fatty acids through cyclization reaction in the cells, and collecting the culture product.

2. The method of claim 1, wherein, The gland cells are derived from the glands of Moschus spp. animals or Ondatra zibethicus animals, preferably from the glands of Moschus berezovskii, Moschus chrysogaster, Moschus moschiferus, Moschus leucogaster, Moschus fuscus, Ondatra zibethicus animals, or from stem cells with multi-differentiation potential.

3. The method of claim 1, wherein, The culture medium comprises cytokines and small molecule components selected from one or more of hormone-like molecules or free fatty acids, preferably the hormone-like molecules are selected from one or more of androgens, prostaglandins, estrogens; more preferably the androgens are selected from dihydrotestosterone or testosterone undecanoate.

4. The method of claim 1, wherein, The culture product comprises one or more of cyclopentadecanone, muscone, cyclohexadecenone, carbonylbicyclo [9, 3, 1] pentadecane. 5.The use of gland cells, or gland cells cultured in 2D or 3D, or gland cells, gland cell clusters or gland organoids with androgen secretion differentiated from adult stem cells or pluripotent stem cells, or mature gland cells, gland cell clusters or gland organoids differentiated in the preparation or synthesis of macrocyclic ketone compounds.

6. Use according to claim 5, characterized in that, The gland cells are derived from the glands of Moschus spp. animals or Ondatra zibethicus animals, or from stem cells with multi-differentiation potential.

7. Use according to claim 5, characterized in that, The use further comprises culturing the gland cells in a culture medium by 2D or 3D culture method, the culture medium comprising cytokines and small molecule components selected from one or more of hormone-like molecules or free fatty acids, preferably the hormone-like molecules are selected from one or more of androgens, prostaglandins, estrogens; more preferably the androgens are selected from dihydrotestosterone or testosterone undecanoate.

8. Use according to claim 5, characterized in that, The use further comprises culturing gland cells in a culture medium by 2D or 3D culture method, adding reaction substrates aldehydes and fatty acids, and biosynthesizing macrocyclic ketone compounds from aldehydes and fatty acids through cyclization reaction in the cells.

9. Use according to claim 8, characterized in that, The macrocyclic ketone compound includes one or more of cyclopentadecanone, muscone, cyclohexadecenone, carbonylbicyclo[9,3,1]pentadecane.

10. A differentiation medium comprising: Cytokines and small molecule components selected from one or more of hormone-like molecules or free fatty acids, preferably the hormone-like molecules are selected from one or more of androgens, prostaglandins, estrogens; more preferably the androgens are selected from dihydrotestosterone or testosterone undecanoate.

11. Use of the differentiation medium of claim 10 for differentiating gland cells, gland cell clusters or glandular organoids, or for preparing or synthesizing macrocyclic ketone compounds.

12. A method of 2D and 3D cell culture of sebaceous tissue comprising: For the extracted gland cells, 2D and 3D cell culture is performed by expansion medium including Wnt3A, R-spondin, Noggin, EGF, FGF, androgens, prostaglandins, free fatty acids; preferably the androgens are selected from dihydrotestosterone or testosterone undecanoate.

13. An expansion medium comprising: Wnt3A, R-spondin, Noggin, EGF, FGF, androgens, prostaglandins, free fatty acids; preferably the androgens are selected from dihydrotestosterone or testosterone undecanoate.

14. Use of the expansion medium of claim 13 for expanding culturing gland cells, gland cell clusters or glandular organoids, or for preparing or synthesizing macrocyclic ketone compounds.

15. The organoid prepared or obtained according to the method of any one of claims 1-4 or 12.

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