Streptomyces with C-S bond stereoselective construction function as well as separation and screening method and application thereof

Abstract

The invention provides streptomyces with a C-S bond stereoselective construction function as well as a separation and screening method and application thereof. In particular, the present invention provides a Streptomyces sp., which comprises a strain having a preservation number of CCTCC (China Center For Type Culture Collection) NO: M2025733. The streptomyces disclosed by the invention can be used as a biocatalyst for catalyzing the 4 '-demethyl epipodophyllotoxin to stereoselectively synthesize the 4 alpha-(5' '-fluorobenzoxazole)-4'-demethyl epipodophyllotoxin.

Description

Technical Field

[0001] This invention relates to a Streptomyces sp., its isolation and screening method, and its applications. Specifically, it relates to a Penicillium purpurogenetic strain with stereoselective CS bond construction function, and the Penicillium purpurogenetic strain as a biocatalyst for the biological construction of thiopodophyllode derivatives C(sp.). 3 Applications of )-S bond stereoselectivity. Background Technology

[0002] Podophyllotoxin compounds, including podophyllotoxin (PTOX) and its derivatives, are a class of aryltetrahydronaphthalene lignans containing five rings (A–E) and four consecutive chiral centers (C1–C4). Podophyllotoxin compounds (such as etoposide) are widely used clinically due to their significant antitumor activity; however, their natural structures suffer from poor water solubility and significant toxic side effects, limiting their clinical application. The 4′-demethylated epipodophyllotoxin ring at position 4 is modified via C(sp...) 3 Thiopodophyllodipine derivatives obtained through stereoselective construction of the C(sp)-S bond generally exhibit good antitumor activity and bioavailability. For thiopodophyllodipine derivatives, their C(sp) 3 The stereoconfiguration of the C(sp)-S bond significantly affects biological activity. Compared to O and N, S, with its larger atomic radius and higher polarizability, exhibits stronger nucleophilicity and dynamic reactivity. 3 The stereoselective construction of α-S bonds is a major scientific problem that urgently needs to be solved in the field of synthetic science.

[0003] Classical chemical methods, represented by nucleophilic substitution and free radicals, and modern catalytic strategies, primarily based on transition metal catalysis, are used to construct C(sp) 3 The stereoselectivity of C(sp) bonds is difficult to control, and side reactions are frequent (ACS Catal 2018; 8(11):9899-9906). For example, C(sp) bonds can be constructed using transition metals such as Pd and Ni. 3 When the sulfur atom forms a 3d-S bond, the strong coordination between the lone pair electrons of the sulfur atom and the 3d orbital of the metal catalyst will irreversibly poison the active center of the metal, leading to catalyst deactivation and stereoselectivity decay (the ee value decreases by more than 20%) (Nat Chem 2024; 16(3):466-475), which seriously restricts the universality and application of this technology.

[0004] Biological methods, leveraging the high stereospecificity of enzymes, utilize natural sulfur-containing compounds C(sp) 3 While exhibiting unique advantages in the construction of C(sp)-S bonds, the substrate specificity of enzymes limits their adaptability to non-natural substrates. Currently, non-natural sulfur-containing compounds C(sp) exhibit unique advantages in the construction of C(sp)-S bonds. 3There are two main strategies for constructing C(sp)-S bonds: one is enzymatic kinetic resolution, which involves first synthesizing a racemic sulfide, and then using enzyme selective catalysis to convert a single enantiomer into a C(sp) bond with a specific R or S configuration. 3 The product is a )-S bond (Angew Chem Int Ed Engl 2022; 1; 61(31):e202202363), but because the other enantiomer cannot be converted, the maximum theoretical yield of this strategy is only 50%, resulting in low raw material utilization; the second is prochiral conversion (Nat Commun 2024; 15(1):8332), which uses a reductase to convert the planar C(sp) bond product into a prochiral bond. 2 ) = S double bond is converted to 3D C(sp 3 While this method can overcome yield limitations, it is only applicable to systems containing specific unsaturated bonds and is difficult to be compatible with the skeletons of complex drugs such as aromatic heterocyclic / fused ring drugs. Summary of the Invention

[0005] One object of the present invention is to provide a stereoselective C(sp) solution. 3 Strains that construct )-S bonds.

[0006] Another object of the present invention is to provide applications of the strain.

[0007] Another object of the present invention is to provide a biological method for constructing thiopodophyllotoxin derivatives C(sp) 3 A method for stereoselectivity of )-S bonds.

[0008] On one hand, the present invention provides a Streptomyces sp., which includes the Streptomyces with accession number CCTCCNO:M2025733.

[0009] The Streptomyces with accession number CCTCC NO:M2025733 was isolated and screened by the inventors during the experiment. In this invention, it is also referred to as Streptomyces sp. TangYJ-O-α. It has been deposited through patent procedures, with the deposit date being April 10, 2025. The depositary institution is the China Center for Type Culture Collection (CCTCC). The address of the depositary institution is Wuhan University, Wuhan, China, 430072, China. The accession number is CCTCC NO:M2025733. The classification and nomenclature are: Streptomyces sp.

[0010] The Streptomyces of the present invention can be used to construct stereoselective C(sp) 3 The )-S bond, wherein the Streptomyces species with accession number CCTCC NO:M2025733 can be used to construct the R configuration of C(sp) bonds, wherein the Streptomyces species can be used to construct the R configuration of C(sp) bonds. 3)-S bond products. In some specific embodiments, the present invention achieves stereospecific coupling between the complex fused-ring skeleton of podophyllotoxin 4'-demethylepiotopotoxin (DMEP) and a non-natural sulfur donor, overcoming the dual challenges of the difficulty in stereoselectivity control in traditional chemical synthesis and the limited adaptability of existing biological methods for substrate construction.

[0011] On the other hand, the present invention also provides a Streptomyces preparation containing the Streptomyces described herein. In some specific embodiments, the preparation is a liquid preparation (i.e., bacterial solution). In some specific embodiments, the preparation is a solid preparation (i.e., bacterial powder). In addition to the Streptomyces with accession number CCTCCNO:M2025733, the preparation of the present invention may also include excipients such as protectants commonly found in traditional bacterial preparations.

[0012] On the other hand, the present invention also provides a Streptomyces ferment, which is a metabolite containing bacteria or having bacteria removed, obtained by fermenting Streptomyces or the bacterial preparation described in the present invention in a culture medium.

[0013] In some specific embodiments, the Streptomyces fermentation product of the present invention is prepared according to the following method:

[0014] The Streptomyces or the bacterial preparation described in this invention are fermented in a culture medium. The culture product is centrifuged, and the supernatant of the fermentation broth is collected to obtain a Streptomyces ferment broth with the bacterial cells removed. Optionally, the Streptomyces ferment broth may be further dried to prepare a powder. The drying may be spray drying at low temperature (e.g., not higher than 60°C, preferably not higher than 50°C) or freeze drying.

[0015] In some specific embodiments, the culture medium for the *Streptomyces* of the present invention is a liquid fermentation medium with a pH of 6.0-8.0, containing 10-20 g / L of carbon source, 3-5 g / L of nitrogen source, and 1-2 g / L of inorganic salts. In some specific embodiments, the culture temperature is 25-45°C, and the culture time is 1-7 days, preferably 24-96 hours.

[0016] On the other hand, the present invention also provides the aforementioned Streptomyces, the aforementioned bacterial preparation, or the aforementioned Streptomyces fermentation product as a biocatalyst for the biological construction of thiopodophyllode derivatives C(sp... 3 Applications of stereoselective synthesis of )-S bonds.

[0017] Streptomyces with accession number CCTCC NO: M2025733 can be used to construct R-configuration C(sp) 3 )-S bond products.

[0018] In a second aspect of the invention, a method is provided for using the aforementioned Streptomyces as a biocatalyst in the biological construction of thiopodophyllode derivatives C(sp...)3 Applications of stereoselective synthesis of )-S bonds.

[0019] In this invention, the podophyllotoxin compounds include podophyllotoxin or its derivatives, for example, see literature CN102757443A or literature "Research Progress in Biosynthesis of Podophyllotoxin and its Derivatives" (Meng Zhen, Yao Tingting, Zhao Wei, Li Hongmei, Tang Yajie, Chinese Journal of Biotechnology, 2026, http: / / journals.im.ac.cn / cjbcn) DOI:10.13345 / j.cjb.210258Jun.25,2021,37(6):2026-2038), including but not limited to etoposide (VP-16), teniposide (VM-26), etoposide phosphate (Etopophos), ammonium etoposide (NK611), ester-based perfluorophenyl etoposide (F11782), p-nitroaniline 4′-demethyletoposide (GL-331), alkylamino 4′-demethyletoposide (TOP-53, NPF), difluoropyranose 4′-demethyletoposide (Adva-27a), aminopyrazine 4′-demethyletoposide (QS-ZYX-1-61), deoxypodophyllotoxin (DPT), polyamine 4′-demethyletoposide (F14512), ester-based dioxopentane 4′-demethyletoposide (CAP) 7.1), 5-fluorobenzoxazole / thiazolinitoxin (5F-Bo / Bth-DMEP), 5-fluorobenzoxazole / thiazolinitoxin (5F-Bo / Bth-VM-26), aminoindole / indazole podophyllotoxin (6-IA / 5-ID-PTOX), etc. In this invention, the podophyllotoxin derivative refers to one or more of the following: C(sp... 3 Podophyllotoxin compounds with )-S bonds, or thiopodophyllotoxin derivatives.

[0020] In this invention, the contents of literature CN102757443A and literature "Research progress in biosynthesis of podophyllotoxin and its derivatives" (Meng Zhen, Yao Tingting, Zhao Wei, Li Hongmei, Tang Yajie, Chinese Journal of Biotechnology, 2026, http: / / journals.im.ac.cn / cjbcn DOI:10.13345 / j.cjb.210258Jun.25,2021,37(6):2026-2038) are incorporated herein by reference.

[0021] In some specific embodiments of the present invention, Streptomyces with accession number CCTCC NO:M2025733 is used to catalyze the stereoselective synthesis of 4α-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin from 4′-demethylepiotatoxin.

[0022] In some specific embodiments of the present invention, the 4α-(5″-fluorobenzoxazole)-4′-demethylepiotazotoxin can be used to prepare antitumor drugs.

[0023] On the other hand, the present invention also provides a method for constructing thiopodophyllotoxin derivatives C(sp) using a Streptomyces biological method. 3 A method for stereoselectivity of α-S bonds, the method comprising:

[0024] Using the Streptomyces strain, the bacterial preparation, or the Streptomyces fermentation product described in this invention as a biocatalyst, an R-configuration C(sp) group is constructed. 3 )-S bond thiopodophyllotoxin derivatives.

[0025] In some specific embodiments of the present invention, the *Streptomyces* strain, the bacterial preparation, or the *Streptomyces* fermentation product described herein are used as biocatalysts to catalyze the stereoselective synthesis of 4α-(5″-fluorobenzoxazole)-4′-demethylpodophyllotoxin from 4′-demethylpodophyllotoxin. Specifically, 4′-demethylpodophyllotoxin and 5-fluorobenzoxazole undergo a coupling reaction via a CS bond to stereoselectively synthesize 4α-(5″-fluorobenzoxazole)-4′-demethylpodophyllotoxin.

[0026] In some specific embodiments of the present invention, the *Streptomyces* strain, the bacterial preparation, or the *Streptomyces* fermentation product described herein are used as biocatalysts to catalyze the stereoselective synthesis of 4α-(5″-fluorobenzoxazole)-4′-demethylpodophyllotoxin from 4′-demethylpodophyllotoxin. The reaction substrate may include 4′-demethylpodophyllotoxin, or a mixture of 4′-demethylpodophyllotoxin and 5-fluorobenzoxazole. When the reaction substrate includes 5-fluorobenzoxazole, the amount of 5-fluorobenzoxazole added may be 0-5 times the molar amount of 4′-demethylpodophyllotoxin; for example, the molar ratio of 4′-demethylpodophyllotoxin to 5-fluorobenzoxazole is 1:1 to 1:5.

[0027] In some specific embodiments of the present invention, the thiopodophylloxera derivative C(sp) is constructed using the Streptomyces biomethionage method with accession number CCTCC NO:M2025733. 3The method for stereoselectivity of the 4'-S bond includes the following steps: (1) adding 4'-noreppodophyllotoxin to a liquid fermentation medium and inoculating it with Streptomyces with preservation number CCTCC NO:M2025733 for fermentation culture; (2) after fermentation culture for a period of time (e.g., 1-3 days), adding 5-fluorobenzoxazole and continuing fermentation culture (e.g., 1-7 days) to obtain the biotransformation product. Through numerous experiments using the above method, this invention has found that different endophytic fungi of podophyllobe plants exhibit significant differences in transformation effects. Streptomyces sp. TangYJ-O-α of this invention, compared to other endophytic fungi of podophyllobe plants, can synthesize 4α-(5″-fluorobenzoxazole)-4'-noreppodophyllotoxin with high stereoselectivity.

[0028] The cultivation process of *Streptomyces sp.* TangYJ-O-α described in this invention can refer to various conventional cultivation methods, including slant culture, liquid seed culture, and fermentation culture. The various culture media and conditions used are conventional in the art and are well known to those skilled in the art. In some specific embodiments of this invention, the cultivation of *Streptomyces sp.* TangYJ-O-α includes: inoculating *Streptomyces sp.* TangYJ-O-α into a suitable culture medium, culturing at a suitable temperature and shaking speed to obtain a seed culture. The culture medium formulation may include: glucose 10-50 g / L, yeast extract 5-20 g / L, potassium nitrate 1-5 g / L, magnesium sulfate 0.5-2 g / L, dipotassium hydrogen phosphate 1-3 g / L, and pH 6-8. The cultivation temperature is 28-32℃, the shaking speed is 150-200 rpm, and the cultivation time is 2-3 days.

[0029] In some specific embodiments of the present invention, the thiopodophylloxera derivative C(sp) is constructed using the Streptomyces biomethionage method with accession number CCTCC NO:M2025733. 3 The method for stereoselectivity of the 4α-S bond includes reacting a system containing 4′-noreppodophyllotoxin, 5-fluorobenzoxazole, and Streptomyces or its fermentation broth with accession number CCTCC NO:M2025733 at 25-45°C to obtain 4α-(5″-fluorobenzoxazole)-4′-noreppodophyllotoxin.

[0030] In some specific embodiments of the present invention, the thiopodophylloxera derivative C(sp) is constructed using the Streptomyces biomethionage method with accession number CCTCC NO:M2025733. 3The method for stereoselectivity of the 5'-S bond includes reacting a system containing 4'-noreppodophyllotoxin and Streptomyces with accession number CCTCC NO:M2025733 at 25-45°C to obtain 4α-(5″-fluorobenzoxazole)-4'-noreppodophyllotoxin. The present invention found that fermentation using Streptomyces with accession number CCTCC NO:M2025733, even with a substrate containing 4'-noreppodophyllotoxin and without the addition of 5-fluorobenzoxazole, yields a large amount of 4α-(5″-fluorobenzoxazole)-4'-noreppodophyllotoxin, suggesting that Streptomyces with accession number CCTCC NO:M2025733 can produce 5-fluorobenzoxazole during fermentation.

[0031] According to a specific embodiment of the present invention, the present invention utilizes a Streptomyces biological method to construct thiopodophyllode derivatives C(sp... 3 For stereoselectivity of the )-S bond, the reaction conditions are pH 6.0-8.0, temperature 25-45℃, and reaction time 1-7 days, preferably 24-72 hours.

[0032] According to some specific embodiments of the present invention, the present invention utilizes a Streptomyces biological method to construct thiopodophyllode derivatives C(sp... 3 For stereoselectivity of the )-S bond, the *Streptomyces* strain or its preparation described in this invention is used as a biocatalyst, and the reaction is carried out in a culture medium. The culture medium is a liquid fermentation medium with a pH of 6.5-7.5, containing 10-20 g / L of carbon source, 3-5 g / L of nitrogen source, and 1-2 g / L of inorganic salts. More specifically, the thiopodophyllotoxin derivative C(sp) is constructed using a *Streptomyces* biological method. 3 For stereoselectivity of the )-S bond, add reaction raw materials such as 4′-demethylepiotatoxin (DMEP) and 5-fluorobenzoxazole (5F-Bo) to the fermentation medium, with final concentrations of 0.1-0.5 g / L and 0.05-0.2 g / L, respectively. Inoculate with Streptomyces with preservation number CCTCC NO:M2025733, with a seed culture inoculation amount of 5%-10%, and culture on a shaker at 25-35℃ and 120-180 rpm for 1-7 days.

[0033] In some specific embodiments of the present invention, the present invention utilizes Streptomyces to precisely construct stereoselective C(sp) 3 The 4'-S bond enables stereospecific coupling between the complex fused-ring skeleton of 4'-demethylepiotatoxin (DMEP) and a non-natural sulfur donor, overcoming the dual challenges of the difficulty in stereoselectivity control in traditional chemical synthesis and the limited adaptability of existing biological methods for substrate construction. This provides theoretical and technical support for the design of highly selective biocatalysts and the creation of chiral sulfur-containing drugs.

[0034] In summary, this invention provides a Streptomyces strain and its application, which can act as a biocatalyst to catalyze the stereoselective synthesis of 4α-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin via a CS bond coupling reaction between 4′-demethylepiotatoxin and 5-fluorobenzoxazole.

[0035] This invention is the first to construct C(sp) using a biological method. 3 The )-S bond enables stereospecific coupling between complex fused-ring skeletons and non-natural sulfur donors. This overcomes the difficulty of stereoselectivity control in traditional chemical synthesis and avoids obtaining large mixtures of isomers. At the same time, it solves the problem of limited substrate adaptability in existing biological methods, enabling efficient and stereoselective synthesis of target products. This provides a new technical means for the synthesis of thiopodophyllode derivatives and the development of related antitumor drugs. Attached Figure Description

[0036] Figure 1 Phylogenetic tree of Streptomyces sp. TangYJ-O-α.

[0037] Figure 2 Biotransformation route for the stereoselective synthesis of 4α-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin from Streptomyces sp. TangYJ-O-α.

[0038] Figure 3 The product prepared in Example 2 1 H-NMR spectrum.

[0039] Figure 4 The product prepared in Example 2 13 C-NMR spectrum.

[0040] Figure 5 The image shows the liquid chromatogram of the product prepared in Example 3.

[0041] Preservation of biological materials for patent procedures:

[0042] Streptomyces sp. TangYJ-O-α:

[0043] Deposit date: April 10, 2025;

[0044] Preservation institution: China Center for Type Culture Collection (CCTCC);

[0045] Address of the depository: Wuhan University, Wuhan, China, 430072, China;

[0046] Accession number: CCTCC NO:M2025733;

[0047] Classification and nomenclature: Streptomyces sp. Detailed Implementation

[0048] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0049] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, the two endpoints of each numerical range and any value between the two endpoints may be selected.

[0050] In addition to the specific methods, equipment, and reagents used in the embodiments, based on the knowledge of those skilled in the art and the description of this invention, any prior art methods, equipment, and materials similar to or equivalent to those described, equipment, and reagents in the embodiments of this invention can be used to implement this invention.

[0051] Unless otherwise stated, the experimental methods, detection methods and preparation methods disclosed in this invention all adopt conventional techniques in this technical field.

[0052] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art.

[0053] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment / implementation, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0054] Unless otherwise explicitly stated in the context, as used herein, the singular forms “a / an” and “the” include a plural indicator. That is, the articles “a / an” and “the” are used herein to refer to one or more (i.e., at least one) grammatical object of the article.

[0055] Unless otherwise stated, the terms "podophyllotoxins" and "podophyllotoxins and their derivatives" are used interchangeably and refer to a class of aryltetrahydronaphthalene lignans containing five rings (A–E) and four consecutive chiral centers (C1–C4).

[0056] Unless otherwise stated, the term "podophyllode derivative" refers to derivatives of podophyllode compounds, and more specifically in this invention refers to sulfur-substituted podophyllode compounds, i.e., those having C(sp...) 3 Podophyllotoxin-like compounds with )-S bonds.

[0057] Example 1: Screening and Identification of Streptomyces

[0058] In this embodiment, a Streptomyces strain was isolated from the stems of *Podophyllum hexandrum* collected from Maolan National Nature Reserve, Libo County, Guizhou Province. The isolation method employed was aseptically performed by tearing open the *Podophyllum hexandrum* rhizomes and spreading them evenly on potato agar plates. After enrichment culture at 28°C, liquid culture, streak plating, and single colony picking onto potato agar slants were performed sequentially to obtain a pure culture. The specific method is as follows:

[0059] Take the stems of the Podophyllum hexandrum plant collected from Maolan National Nature Reserve in Libo County, Guizhou Province, cut them into appropriately sized small pieces, place them in 50mL sterile centrifuge tubes, and ultrasonically clean them 3-5 times with sterile water until the sterile water is clear and transparent.

[0060] The stems, after being washed with sterile water, were washed with 0.05% (v / v) Tween 20 for 1 min, followed by rinsing twice with sterile water. Next, they were rinsed with 95% (v / v) ethanol for 6 min, immersed in 5% (v / v) NaClO solution for 6 min, and then rinsed with 75% (v / v) ethanol for 6 min. Finally, they were washed 3-5 times with sterile water to remove chemical residues (the sterile water from the final rinse served as a control; after incubation at 28°C, no sterile growth was observed, demonstrating adequate sterilization).

[0061] Remove the processed stems, leaves, and fruits, and blot them dry with sterile filter paper.

[0062] Sectioning: The stem is longitudinally cut in the center, and the phloem of the longitudinal section is aseptically removed; the leaves are cut into 0.5cm×0.5cm pieces to expose fresh cut surfaces; the samples are mounted on culture medium, PDA medium, and MS medium (stem longitudinal section facing down), and incubated at 28℃. Colonies that grow are picked in time and transferred to new culture medium for streak purification.

[0063] Grinding process: Grind the stems in a sterilized mortar, add an appropriate amount of sterile water, and collect the grinding liquid. The grinding liquid is then divided into 10... -1 , 10 -2Dilute and spread on the culture medium; grind the residue onto the culture medium, PDA medium, and MS medium, place in an incubator at 28°C, and promptly pick out the growing colonies and transfer them to a new culture medium for streak purification.

[0064] ①PDA culture medium (g / L):

[0065]

[0066] ②MS medium (g / L):

[0067]

[0068] ③ Culture medium for bacterial separation (g / L):

[0069]

[0070] Identification of strains

[0071] SDS method for extracting actinomycete genome:

[0072] Isopropanol was pre-cooled at -20°C and ddH2O was preheated at 7°C.

[0073] ① Use a sterile toothpick to pick up spores and inoculate them into TSBY liquid medium, and incubate at 30℃ and 220rpm for 2-4 days;

[0074] ② Take 1.5 mL of bacterial culture into a 2 mL EP tube, centrifuge at 13000 rpm for 1 min, and discard the supernatant;

[0075] ③ Add 500 μL of lysozyme buffer, mix well, centrifuge at 13000 rpm for 1 min, and discard the supernatant;

[0076] ④ Prepare a 2-4 mg / mL lysozyme solution using lysozyme buffer, add 500 μL of suspended bacterial cells, mix thoroughly, and incubate at 37°C for 30 min, inverting the solution every 5-10 min to mix.

[0077] ⑤ Add 350 μL of 6% SDS (an anionic surfactant that can dissolve cell membrane and nuclear membrane proteins, causing cell membrane and nuclear membrane to rupture), mix well, and then incubate in a water bath at 37°C until the solution becomes clear and transparent;

[0078] ⑥ Add 1 / 10 volume of 3M NaAc (pH 5.2) to neutralize the pH, add 250 μL of phenol / chloroform / isoamyl alcohol 25:24:1, mix thoroughly by inverting until emulsion is formed (do not shake), centrifuge at 13000 rpm for 10 min, and transfer the supernatant to a new 1.5 mL EP tube;

[0079] ⑦ Add an equal volume of isopropanol, gently invert to mix, a white filamentous precipitate will form (DNA precipitation). If there is not much precipitate, place at -20℃ for 1-2 hours to promote DNA precipitation; centrifuge at 13000 rpm for 20 minutes and discard the supernatant.

[0080] ⑧ Wash the DNA precipitate twice with 500 μL of 70% ethanol, centrifuge at 13000 rpm for 5 min, remove the ethanol after washing, and centrifuge once with an empty tube.

[0081] ⑨ Open the lid at room temperature and dry slightly. Add an appropriate amount (20-40 μL) of sterile ddH2O to dissolve the DNA (DNA re-dissolves slowly, so incubate at 37°C for 30 min). Store at -20°C.

[0082] The extracted genome was amplified by PCR using universal primers 27F and 1492R. The PCR system consisted of: 25 μL 2xTaqPlus PCR Master Mix, 19 μL ddH2O, 2 μL universal primer 27F, 2 μL universal primer 1492R, and 2 μL template DNA. 10 μL of the PCR product was electrophoresed on a 1.5% agarose gel. After confirming the presence of clear and bright bands, the remaining PCR product was sent to a sequencing company for sequencing. The sequencing results are shown in lcl|Query_290197 (SEQ ID NO:1).

[0083] The sequencing results were compared and analyzed with gene sequences from the NCBI database using the Blast search program, and a phylogenetic tree was constructed. Figure 1 (As shown). The strain TangYJ-O-α was found to be most homologous to the type strain Streptomyces atroolivaceusstrain NBRC 12741, thus identifying TangYJ-O-α as belonging to the genus Streptomyces, and naming it Streptomyces sp. TangYJ-O-α. This strain has been deposited through patent procedures, with the deposit date being April 10, 2025; depositary institution: China Center for Type Culture Collection (CCTCC); address: Wuhan University, Wuhan, China, 430072, China; accession number: CCTCC NO: M2025733; classification and nomenclature: Streptomyces sp.

[0084] lcl|Query_290197(SEQ ID NO:1)

[0085] ACCATGCAAGTCGAACGATGAAGCCCTTCGGGGTGGATTAGTGGCGAACGGGT

[0086] GAGTAACACGTGGGCAATCTGCCCTTCACTCTGGGACAAGCCCTGGAAACGGG

[0087] GTCTAATACCGGATAACACTCTGTCCCTCATGGGGCGGGGTTAAAAGCTCCGGC

[0088] GGTGAAGGATGAGCCCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCA

[0089] AGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGA

[0090] GACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGG

[0091] CGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAA

[0092] ACCTCTTTCAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGC

[0093] CGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCG

[0094] GAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGATGTGAAAGC

[0095] TCGGGGCTTAACCCCGAGTCTGCATTCGATACGGGCTAGCTAGAGTGTGGTAGG

[0096] GGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACA

[0097] CCGGTGGCGAAGGCGGATCTCTGGGCCATTACTGACGCTGAGGAGCGAAAGCG

[0098] TGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGTTGGGA

[0099] ACTAGGTGTTGGCGACATTCCACGTCGTCGGTGCCGCAGCTAACGCATTAAGTT

[0100] CCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGG

[0101] CCCGCACAAGCAGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTT

[0102] ACCAAGGCTTGACATATACCGGAAAGCATCAGAGATGGTGCCCCCCTTGTGGTC

[0103] GGTATACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTA

[0104] AGTCCCGCAACGAGCGCAACCCTTGTTCTGTGTTGCCAGCATGCCCTTCGGGGT

[0105] GATGGGGACTCACAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACG

[0106] ACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCC

[0107] GGTACAATGAGCTGCGATGCCGCGAGGCGGAGCGAATCTCAAAAAGCCGGTCT

[0108] CAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTTGCTAGTAAT

[0109] CGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCC

[0110] GTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGCCAACCCCTTGTGGGAGGGAGCTGTCG.

[0111] Example 2: Synthesis of 4α-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin using Streptomyces with accession number CCTCC NO:M2025733

[0112] This embodiment provides a biological method for constructing thiopodophyllotoxin derivative C(sp) using Streptomyces p. TangYJ-O-α (accession number CCTCC NO: M2025733). 3 4α-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin (product I) was synthesized by a stereoselective method involving the 5″-S bond.

[0113] 4′-Desmethylepiotatoxin: purchased from Xi'an Helin Bioengineering Co., Ltd., purity 98%;

[0114] 5-Fluorobenzoxazole (5F-Bo): Purchased from Bid Pharmaceutical, purity 98%.

[0115] Strain culture: Streptomyces sp. TangYJ-O-α was inoculated into a suitable culture medium and cultured at a suitable temperature and shaking speed to obtain a seed culture.

[0116] Specifically, the Streptomyces sp. culture process is as follows:

[0117] (1) Slant culture: Culture medium: 20% potato, 2% glucose, 2% agar, pH 7 (the content of each component is a weight-volume percentage, i.e. g / 100 ml, the same below), sterilize at 115℃ for 30 minutes, cool after sterilization, prepare slant, inoculate with Streptomyces strain, and incubate at 28℃ for 2-4 days.

[0118] (2) Liquid seed culture: Culture medium: 3% tryptone soybean broth, 0.1% yeast powder, 0.1% peptone, pH 7, add appropriate amount of 3-4mm glass beads, liquid volume 50 ml / 250 ml shake flask, sterilize at 121℃ for 20 minutes, cool after sterilization, inoculate with slant inoculum, culture at 28℃, 200 rpm for 1-2 days as liquid seed;

[0119] (3) Transformation culture: Fermentation medium: 0.5% yeast powder, 0.05% dipotassium hydrogen phosphate, 0.05% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.05% sodium chloride, 0.01% ferrous sulfate, pH value of 7, liquid volume of 50 ml / 250 ml shake flask, sterilized at 121℃ for 20 minutes, cooled after sterilization, inoculated with Streptomyces liquid seed with preservation number CCTCC NO:M2025733, inoculation amount of 5%, cultured at 28℃, 200 rpm for 13 hours.

[0120] Biotransformation: 4′-demethylepiotazotoxin (DMEP) was added to the fermentation medium to a final concentration of 0.3 g / L, followed by the addition of 5-fluorobenzoxazole (5F-Bo) to a final concentration of 0.1 g / L. The reaction was carried out for 5 days at 28-32°C and 150-200 rpm on a shaker.

[0121] Product separation and identification: After the reaction, the fermentation broth was centrifuged, and the supernatant was collected. Liquid-liquid extraction was used, with the supernatant extracted three times with ethyl acetate. The organic phases were combined and concentrated by rotary evaporation. Then, separation, purification, and identification were performed by silica gel column chromatography and high-performance liquid chromatography. A petroleum ether-ethyl acetate mixture with a volume ratio of 10-20:1 was used as the eluent for silica gel column chromatography. Identification revealed 4α-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin (product I), with a diastereomeric excess value (de>99%).

[0122] Figure 2 The diagram shows the biotransformation route for the stereoselective synthesis of 4α-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin by Streptomyces.

[0123] Figure 3 The product prepared as described above in this embodiment 1 H-NMR. Figure 4 The product prepared as described above in this embodiment 13 C-NMR.

[0124] from Figure 3 and Figure 4 As can be seen from this embodiment, 4α-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin was successfully prepared.

[0125] Seed fermentation broth of *Streptomyces p.* (CCTCC NO: M2025733) was prepared using the same method described above. Fermentation was carried out for 2 days, and the supernatant was collected by centrifugation to obtain *Streptomyces p.* fermentation product with cell removal. Biotransformation: 5% (v / v) of the cell-removed *Streptomyces p.* fermentation supernatant (CCTCC NO: M2025733) was added to a reaction system containing 0.3 g / L of 4′-demethylepiotatoxin (DMEP) and 0.1 g / L of 5-fluorobenzoxazole (5F-Bo). The reaction was carried out at 30°C for 3 days. The reaction product was purified and identified. 1 H-NMR and 13 The C-NMR spectrum shows that 4α-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin was successfully prepared.

[0126] Example 3: Synthesis of 4α-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin using Streptomyces with accession number CCTCC NO:M2025733

[0127] In this embodiment, the culture process of Streptomyces with accession number CCTCC NO:M2025733 is as follows:

[0128] (1) Slant culture: Culture medium: potato 15%, glucose 2%, agar 2%, pH 7 (the content of each component is a weight-volume percentage, i.e. g / 100 ml, the same below), sterilize at 115℃ for 30 minutes, cool after sterilization, prepare slant, inoculate with Streptomyces strain, and incubate at 28℃ for 3 days.

[0129] (2) Liquid seed culture: Culture medium: 2.5% tryptone soybean broth, 0.5% yeast powder, 0.2% peptone, pH 7, add appropriate amount of 3-4mm glass beads, liquid volume 50 ml / 250 ml shake flask, sterilize at 121℃ for 20 minutes, cool after sterilization, inoculate with slant culture, culture at 28℃ and 200 rpm for 1 day as liquid seed;

[0130] (3) Transformation culture: Fermentation medium: 0.8% yeast powder, 0.05% dipotassium hydrogen phosphate, 0.05% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.05% sodium chloride, 0.01% ferrous sulfate, pH value of 7, liquid volume of 50 ml / 250 ml shake flask, sterilized at 121℃ for 20 minutes, cooled after sterilization, inoculated with Streptomyces liquid seed with preservation number CCTCC NO:M2025733, inoculation amount of 5%, cultured at 28℃ and 200 rpm for 24 hours.

[0131] Transformation culture: The reaction substrate was added to the fermentation medium containing *Streptomyces* strain with preservation accession number CCTCC NO: M2025733, and the mixture was incubated at 30°C and 150 rpm for 3 days. In this example, four experimental groups were set up: Group i, no reaction substrate added; Group ii, 4′-demethylepiotatoxin (DMEP) added to a final concentration of 0.3 g / L in the fermentation medium; Group iii, 5-fluorobenzoxazole (5F-Bo) added to a final concentration of 0.1 g / L in the fermentation medium; Group iv, 4′-demethylepiotatoxin (DMEP) added to a final concentration of 0.3 g / L in the fermentation medium, and 5-fluorobenzoxazole (5F-Bo) added to a final concentration of 0.1 g / L in the fermentation medium.

[0132] Product separation and identification: After the reaction, the fermentation broth was centrifuged, and the supernatant was collected. The supernatant was extracted three times with ethyl acetate using a liquid-liquid extraction method. The organic phases were combined and concentrated by rotary evaporation. Then, separation, purification, and identification were performed by silica gel column chromatography and high-performance liquid chromatography. A petroleum ether-ethyl acetate mixture with a volume ratio of 10-20:1 was used as the eluent for silica gel column chromatography.

[0133] See the liquid chromatograms of the products from each experimental group. Figure 5 Group v was a standard control of 4α-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin, and group vi was a standard control of 4β-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin. As can be seen from the figure, this embodiment successfully prepared a large quantity of 4α-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin without the addition of 5-fluorobenzoxazole to the reaction substrate.

[0134] Example 4: Detection of the antitumor activity of the product

[0135] Experimental materials:

[0136] Example 2: Product I prepared by catalysis from Streptomyces sp. TangYJ-O-α seed culture;

[0137] Control compounds (such as the known antitumor drug etoposide);

[0138] Tumor cell lines (such as HeLa, HepG2, etc.).

[0139] Experimental Methods: The MTT assay was used to detect the inhibitory activity of the product on tumor cells. Tumor cells in logarithmic growth phase were adjusted to the appropriate concentration and seeded into 96-well plates. After a period of culture, different concentrations of product I and the control compound were added. After further culture for a certain period, MTT solution was added, and after incubation, DMSO was added to dissolve and crystallize the cells. The absorbance was measured, and the cell inhibition rate was calculated.

[0140] The test results of the antitumor activity of product I are shown in Table 1.

[0141] The results showed that the product I of the present invention had a significant growth inhibitory effect on tumor cell lines such as Hela and HepG2, indicating that it has potential anti-tumor application value.

[0142] Table 1. Antitumor activity of Product I

[0143]

[0144] a IC 50 The value is the average of three repeated measurements.

[0145] The above embodiments are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention to achieve the same purpose. Therefore, all equivalent technical solutions also fall within the protection scope of this invention.

Claims

1. A Streptomyces sp. comprising the Streptomyces with accession number CCTCC NO:M2025733.

2. A Streptomyces preparation containing the Streptomyces as described in claim 1.

3. A Streptomyces fermentation product, which is a metabolite containing bacterial cells or after removing bacterial cells, obtained by fermenting Streptomyces of claim 1 or the bacterial preparation of claim 2 in a culture medium; Preferably, the Streptomyces ferment is prepared according to the following method: The Streptomyces of claim 1 or the bacterial preparation of claim 2 is fermented in a culture medium. The culture product is centrifuged and the supernatant of the fermentation broth is collected to obtain Streptomyces fermentation product with the bacterial cells removed. Optionally, the Streptomyces fermentation product is further dried to prepare a powder. More preferably, the culture medium is a liquid fermentation medium with a pH of 6.0-8.0, containing 10-20 g / L of carbon source, 3-5 g / L of nitrogen source, and 1-2 g / L of inorganic salt; More preferably, the culture temperature is 25-45℃, and the culture time is 1-7 days, more preferably 24-96 hours.

4. The Streptomyces of claim 1, the microbial preparation of claim 2, or the Streptomyces fermentation product of claim 3 as a biocatalyst for the biological construction of thiopodophyllode derivatives C(sp...) 3 Applications of stereoselective synthesis of )-S bonds.

5. The application according to claim 4, wherein, Streptomyces with accession number CCTCC NO:M2025733 was used to catalyze the stereoselective synthesis of 4α-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin from 4′-demethylepiotatoxin.

6. The application according to claim 5, wherein, The 4α-(5″-fluorobenzoxazole)-4′-demethylepiotazotoxin is used in the preparation of antitumor drugs.

7. A method for constructing thiopodophyllotoxin derivatives C(sp) using Streptomyces biosynthesis 3 A method for stereoselectivity of α-S bonds, the method comprising: Using the Streptomyces of claim 1, the microbial preparation of claim 2, or the Streptomyces ferment of claim 3 as a biocatalyst, construct an R-configuration C(sp) group. 3 Thiopodophyllodes derivatives with )-S bonds.

8. The method according to claim 7, wherein, The Streptomyces of claim 1, the bacterial preparation of claim 2, or the Streptomyces ferment of claim 3 are used as biocatalysts to catalyze the stereoselective synthesis of 4α-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin from 4′-demethylepiotatoxin.

9. The method according to claim 8, wherein, The reaction conditions are pH 6.0-8.0, temperature 25-45℃, and reaction time 1-7 days, preferably 24-72 hours; Preferably, the reaction substrate includes 4′-demethylepiotopotoxin, or a mixture of 4′-demethylepiotopotoxin and 5-fluorobenzoxazole; the amount of 5-fluorobenzoxazole added can be 0-5 times the molar amount of 4′-demethylepiotopotoxin, for example, the molar ratio of 4′-demethylepiotopotoxin to 5-fluorobenzoxazole is 1:1-1:

5.

10. The method according to claim 8 or 9, wherein, The biocatalyst used in the reaction is the Streptomyces as described in claim 1 or the bacterial preparation as described in claim 2. The reaction is carried out in a culture medium, which is a liquid fermentation medium with a pH of 6.0-8.0, containing 10-20 g / L of carbon source, 3-5 g / L of nitrogen source, and 1-2 g / L of inorganic salt.

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