Penicillium purpurogenum with C-S bond stereoselective construction function as well as domestication method and application of penicillium purpurogenum

Abstract

The invention provides penicillium purpurogenum with a C-S bond stereoselective construction function as well as a domestication method and application of the penicillium purpurogenum. In particular, the present invention provides a Penicillium purpurogenum, which comprises a strain having a preservation number of CCTCC (China Center For Type Culture Collection) NO: M2025735 and / or a strain having a preservation number of CCTCC NO: M2025734. The penicillium purpurogenum provided by the invention can be used as a biocatalyst to catalyze a coupling reaction of 4 '-demethylated epipodophyllotoxin and 5-fluorobenzoxazole through a C-S bond, and 4 alpha-(5' '-fluorobenzoxazole)-4'-demethylated epipodophyllotoxin or 4 beta-(5 ''-fluorobenzoxazole)-4 '-demethylated epipodophyllotoxin is synthesized in a stereoselective manner.

Description

Technical Field

[0001] This invention relates to a *Penicillium purpurogenum* fungus, its domestication method, and its applications. Specifically, it relates to a *Penicillium purpurogenum* fungus with stereoselective CS bond construction capabilities, its domestication method, and the use of the *Penicillium purpurogenum* fungus as a biocatalyst in the biological construction of thiopodophyllode derivatives C(sp...)... 3 Applications of )-S bond stereoselectivity. Background Art

[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 )-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.

[0005] CN102757443A discloses a sulfur-substituted podophyllotoxin derivative and its biotransformation, separation, and purification method. The method involves using endophytic fungi of podophyllotoxin plants (e.g., *Penicillium purpurogenum* YJTang, accession number CCTCC NO. M2010262) to modify podophyllotoxin or its derivatives to obtain sulfur-substituted podophyllotoxin derivatives. However, this strain cannot achieve stereoselective synthesis of 4α-(5″-fluorobenzoxazole)-4′-demethylepiophyllotoxin or 4β-(5″-fluorobenzoxazole)-4′-demethylepiophyllotoxin. Summary of the Invention

[0006] One object of the present invention is to provide a stereoselective C(sp) solution. 3 Strains that construct )-S bonds.

[0007] Another object of the present invention is to provide applications of the strain.

[0008] 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.

[0009] On one hand, the present invention provides a purpurogenum fungus, including the purpurogenum fungus with accession number CCTCC NO:M2025735 and / or the purpurogenum fungus with accession number CCTCC NO:M2025734.

[0010] The *Penicillium purpurogenum* with accession number CCTCC NO: M2025735 was obtained by the inventors of this invention through mutagenesis screening during the experimental process. In this invention, it is also referred to as *Penicillium purpurogenum* 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: M2025735. The classification and nomenclature are *Penicillium purpurogenum*.

[0011] The *Penicillium purpurogenum* with accession number CCTCC NO: M2025734 was obtained by the inventors of this invention through mutagenesis screening during the experimental process. In this invention, it is also referred to as *Penicillium purpurogenum* TangYJ-O-β. 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: M2025734. The classification and nomenclature are *Penicillium purpurogenum*.

[0012] The Penicillium purpureum of this invention can be used to construct stereoselective C(sp) 3 The -S bond, wherein the *Penicillium purpureus* species with accession number CCTCCNO:M2025735 can be used to construct the R configuration of C(sp) bonds, wherein the R configuration of C(sp) bonds can be used to construct the R configuration of C(sp) bonds. 3 The s-S bond product, *Penicillium purpureoides* with accession number CCTCC NO: M2025734, can be used to construct S-configured 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.

[0013] On the other hand, the present invention also provides a Penicillium purpureum fungal preparation containing the Penicillium purpureum fungus described herein. In some specific embodiments, the fungal preparation is a liquid preparation (i.e., fungal solution). In some specific embodiments, the fungal preparation is a solid preparation (i.e., fungal powder). In addition to the Penicillium purpureum fungus with accession number CCTCC NO:M2025735 and / or the Penicillium purpureum fungus with accession number CCTCC NO:M2025734, the fungal preparation of the present invention may also include excipients such as protectants commonly found in traditional fungal preparations.

[0014] On the other hand, the present invention also provides a Penicillium purpureum ferment, which is a metabolite containing bacterial cells or after removing bacterial cells obtained by fermenting Penicillium purpureum or the bacterial preparation described in the present invention in a culture medium.

[0015] In some specific embodiments, the Penicillium purpureum ferment broth of the present invention is prepared according to the following method:

[0016] The *Penicillium purpureum* or the *Penicillium purpureum* preparation described in this invention is fermented in a culture medium. The culture product is centrifuged, and the supernatant of the fermentation broth is collected to obtain *Penicillium purpureum* fermented product with the bacterial cells removed. Optionally, the *Penicillium purpureum* fermented product may be further dried to prepare a powder. The drying may be spray drying under low temperature conditions (e.g., not higher than 60°C, preferably not higher than 50°C) or freeze drying.

[0017] In some specific embodiments, the culture medium for the *Penicillium purpureus* of the present invention 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. In some specific embodiments, the culture temperature is 25-45°C, and the culture time is 1-7 days.

[0018] On the other hand, the present invention also provides the aforementioned *Penicillium purpureum*, the aforementioned fungal preparation, or the aforementioned *Penicillium purpureum* fermentation product as a biocatalyst for the biological construction of thiopodophyllode derivatives C(sp... 3 Applications of stereoselective synthesis of )-S bonds.

[0019] The *Penicillium purpureum* fungus with accession number CCTCC NO: M2025735 can be used to construct the R configuration of C(sp) 3 )-S bond products. The *Penicillium purpureus* species with accession number CCTCC NO: M2025734 can be used to construct S-configured C(sp) bonds. 3 )-S bond products.

[0020] In a second aspect of the invention, a method is provided for using the aforementioned *Penicillium purpureum* fungus as a biocatalyst in the biological construction of thiopodophyllode derivatives C(sp...) 3 Applications of stereoselective synthesis of )-S bonds.

[0021] 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.

[0022] 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.

[0023] In some specific embodiments of the present invention, the Penicillium purpureus with accession number CCTCC NO:M2025735 is used to catalyze the stereoselective synthesis of 4α-(5″-fluorobenzoxazole)-4′-demethylpetropophyllotoxin from 4′-demethylpetropophyllotoxin and 5-fluorobenzoxazole.

[0024] In some specific embodiments of the present invention, the Penicillium purpureus with accession number CCTCC NO:M2025734 is used to catalyze the stereoselective synthesis of 4β-(5″-fluorobenzoxazole)-4′-demethylpetropophyllotoxin from 4′-demethylpetropophyllotoxin and 5-fluorobenzoxazole.

[0025] In some specific embodiments of the present invention, the 4α-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin or 4β-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin should be used to prepare antitumor drugs.

[0026] On the other hand, the present invention also provides a method for constructing thiopodophyllotoxin derivatives C(sp) using Penicillium purpureum via a biological method. 3 A method for stereoselectivity of α-S bonds, the method comprising:

[0027] Using the *Penicillium purpureum* strain, the bacterial preparation, or the *Penicillium purpureum* fermentation product as a biocatalyst, 4′-demethyleppodophyllotoxin and 5-fluorobenzoxazole are coupled via a CS bond to stereoselectively synthesize 4α-(5″-fluorobenzoxazole)-4′-demethyleppodophyllotoxin or 4β-(5″-fluorobenzoxazole)-4′-demethyleppodophyllotoxin.

[0028] According to a specific embodiment of the present invention, the present invention utilizes a biological method involving *Penicillium purpureum* to construct thiopogonin derivatives C(sp...). 3 For stereoselectivity of the )-S bond, the reaction conditions are pH 6.0-8.0, temperature 25-35℃, reaction time 1-7 days (preferably 24-72 hours), and the molar ratio of substrate 4′-demethylepiotazotoxin to 5-fluorobenzoxazole is 1:1-1:5.

[0029] According to some specific embodiments of the present invention, the present invention utilizes a biological method based on Penicillium purpureus to construct thiopogonin derivatives C(sp... 3 When the )-S bond is stereoselective, the reaction is carried out in a culture medium, namely a liquid fermentation medium with pH 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 biological method with *Penicillium purpureum*. 3For 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 Penicillium purpureiogenum with accession number CCTCC NO:M2025735 or accession number CCTCC NO:M2025734 at an inoculum size of 5%-10%, and culture on a shaker at 25-35℃ and 120-180 rpm.

[0030] In some specific embodiments of the present invention, the present invention utilizes Penicillium purpureum to precisely construct stereoselective C(sp) groups. 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.

[0031] In summary, this invention provides a class of Penicillium purpureum-producing fungi and their applications. These fungi can act as biocatalysts to catalyze the stereoselective synthesis of 4α-(5″-fluorobenzoxazole)-4′-demethylpetropophyllotoxin or 4β-(5″-fluorobenzoxazole)-4′-demethylpetropophyllotoxin via a CS bond coupling reaction.

[0032] 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, overcoming the difficulty of stereoselectivity control in traditional chemical synthesis and avoiding the generation of 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, and providing a new technical means for the synthesis of thiopodophyllotoxin derivatives and the development of related antitumor drugs. Attached Figure Description

[0033] Figure 1 Biotransformation route for the stereoselective synthesis of 4α-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin from the purpurogenum fungus Penicillium purpurogenum TangYJ-O-α.

[0034] Figure 2 The product prepared in Example 2 1 H-NMR spectrum.

[0035] Figure 3 4% of the product prepared in Example 2 13 C-NMR spectrum.

[0036] Figure 4 Biotransformation route for the stereoselective synthesis of 4β-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin from Penicillium purpurogenum TangYJ-O-β.

[0037] Figure 5 The product prepared in Example 3 1 H-NMR spectrum.

[0038] Figure 6 4% of the product prepared in Example 3 13 C-NMR spectrum.

[0039] Preservation of biological materials for patent procedures:

[0040] (a) Penicillium purpurogenum TangYJ-O-α:

[0041] Deposit date: April 10, 2025;

[0042] Preservation institution: China Center for Type Culture Collection (CCTCC);

[0043] Address of the depository: Wuhan University, Wuhan, China, 430072, China;

[0044] Accession number: CCTCC NO:M2025735;

[0045] Classification and nomenclature: Penicillium purpurogenum.

[0046] (ii) Penicillium purpurogenum TangYJ-O-β:

[0047] Deposit date: April 10, 2025;

[0048] Preservation institution: China Center for Type Culture Collection (CCTCC);

[0049] Address of the depository: Wuhan University, Wuhan, China, 430072, China;

[0050] Accession number: CCTCC NO:M2025734;

[0051] Classification and nomenclature: Penicillium purpurogenum. DETAILED DESCRIPTION

[0052] 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.

[0053] 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.

[0054] 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.

[0055] Unless otherwise stated, the experimental methods, detection methods and preparation methods disclosed in this invention all adopt conventional techniques in this technical field.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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).

[0060] 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.

[0061] Example 1: Screening and identification of Penicillium purpureus

[0062] Seventeen strains of *Penicillium purpureum* were purchased from the China General Microbiological Culture Collection Center, the China Agricultural Microbiological Culture Collection Center, the China Forestry Microbiological Culture Collection Center, and the China Type Culture Collection Center. These strains were then induced and trained according to the method described in this invention to screen for strains capable of stereoselectively synthesizing 4α-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin or 4β-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin.

[0063] (1) Culture medium for strain domestication

[0064] The glucose in the liquid culture medium was replaced with different concentrations of 4′-demethylepiotatoxin substrate, and the specific culture media selected were as follows (g / L):

[0065] No. 1: Glucose 30, Sodium nitrate 2, Dipotassium hydrogen phosphate 1, Magnesium sulfate heptahydrate 0.5, Sodium chloride 0.5, Ferrous sulfate heptahydrate 0.01, pH 6.6;

[0066] Item 2: 4′-Demethylepiotatoxin 0.1, Sodium nitrate 2, Dipotassium hydrogen phosphate 1, Magnesium sulfate heptahydrate 0.5, Sodium chloride 0.5, Ferrous sulfate heptahydrate 0.01, pH 6.6;

[0067] No. 3: 4′-demethylepiotatoxin 0.5, sodium nitrate 2, dipotassium hydrogen phosphate 1, magnesium sulfate heptahydrate 0.5, sodium chloride 0.5, ferrous sulfate heptahydrate 0.01, pH 6.6;

[0068] Item 4: 4′-Demethylepiotatoxin 1, Sodium nitrate 2, Dipotassium hydrogen phosphate 1, Magnesium sulfate heptahydrate 0.5, Sodium chloride 0.5, Ferrous sulfate heptahydrate 0.01, pH 6.6;

[0069] Item 5: 4′-demethylepiotatoxin 2, sodium nitrate 2, dipotassium hydrogen phosphate 1, magnesium sulfate heptahydrate 0.5, sodium chloride 0.5, ferrous sulfate heptahydrate 0.01, pH 6.6.

[0070] 4′-Desmethylepiotatoxin: purchased from Xi'an Helin Bioengineering Co., Ltd., purity 98%; 5-Fluorobenzoxazole (5F-Bo): purchased from Bid Pharmaceutical, purity 98%.

[0071] (2) Methods of strain domestication

[0072] Take the PDA slant seed cultured at 28℃ for 4 days, wash it with 10 mL of sterile water, and then inoculate the washed slant seed culture into medium No. 1 and culture at 28℃ and 120 rpm for 4 days.

[0073] Take 1 mL of seed culture from seed culture No. 1, which has been cultured at 28℃ and 120 rpm for 4 days, and inoculate it into medium No. 2. Culture at 28℃ and 120 rpm for 4 days. At the same time, take 1 mL of seed culture No. 1 and spread it onto solid medium No. 2. Culture at 28℃.

[0074] Take 1 mL of seed culture from seed culture No. 2, which has been cultured at 28℃ and 120 rpm for 4 days, and inoculate it into medium No. 3. Culture for 4 days at 28℃ and 120 rpm. At the same time, take 1 mL of seed culture No. 2 and spread it onto solid medium No. 3. Culture for 28℃.

[0075] Take 1 mL of seed culture from seed culture No. 3, which has been cultured at 28℃ and 120 rpm for 4 days, and inoculate it into medium No. 4. Culture at 28℃ and 120 rpm for 4 days. At the same time, take 1 mL of seed culture No. 3 and spread it onto solid medium No. 4. Culture at 28℃.

[0076] Take 1 mL of seed culture from seed culture No. 4, which has been cultured at 28℃ and 120 rpm for 4 days, and inoculate it into medium No. 5. Culture for 4 days at 28℃ and 120 rpm. At the same time, take 1 mL of seed culture No. 4 and spread it onto solid medium No. 5. Culture for 28℃.

[0077] The growth of the above-mentioned Penicillium purpureus on liquid and solid culture media was observed and recorded. The maximum tolerance concentration of the substrate 4′-demethylepiotatoxin was determined based on the growth of the bacteria. Then, a single colony was picked from the plate with the maximum tolerance to the substrate 4′-demethylepiotatoxin, inoculated on PDA slant, and cultured at 28°C to obtain the domesticated pure culture.

[0078] In this invention, after domesticating 17 strains of *Penicillium purpurogenum*, it was found that one of the domesticated strains (self-named *Penicillium purpurogenum* TangYJ-O-α in this invention) could stereoselectively synthesize 4α-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin (product I), with a diastereomeric excess value de>99%. This strain, *Penicillium purpurogenum* TangYJ-O-α, has been deposited at the China Center for Type Culture Collection (CCTCC) (address: Wuhan University, Wuhan, China, 430072, China), deposited on April 10, 2025; accession number: CCTCCNO: M2025735; classification name: *Penicillium purpurogenum*. Another domesticated strain of *Penicillium purpurogenum* (self-named *Penicillium purpurogenum* in this invention)... The strain *Penicillium purpurogenum* TangYJ-O-β can stereoselectively synthesize 4β-(5″-fluorobenzoxazole)-4′-demethylepiotopotoxin (product II) with a diastereomeric excess value (de) ≥ 96%. This strain is deposited at the China Center for Type Culture Collection (CCTCC) (address: Wuhan University, Wuhan, China, 430072, China), dated April 10, 2025; accession number: CCTCC NO: M2025734; classification name: *Penicillium purpurogenum*. Other domesticated strains of *Penicillium* and 17 strains of *Penicillium* before domestication could not stereoselectively synthesize 4α-(5″-fluorobenzoxazole)-4′-demethylepiotopotoxin or 4β-(5″-fluorobenzoxazole)-4′-demethylepiotopotoxin.

[0079] The Penicillium purpureus of this invention, with accession number CCTCC NO:M2025735 or accession number CCTCC NO:M2025734, can be cultured at a temperature of 25-35℃, and can be cultured on slant agar and frozen for preservation. Preferably, the slant culture medium formula is: glucose 20-40 g / L, magnesium sulfate 0.5-1.5 g / L, potassium dihydrogen phosphate 1-2 g / L, thiamine hydrochloride 0.05-0.15 g / L, agar 15-25 g / L, and potato extract can be selectively added (usually 100-300 g of water-boiled potato extract per liter of culture medium).

[0080] According to a preferred embodiment of the present invention, the primary and secondary liquid seed culture medium formulations for *Penicillium purpureus* (accession number CCTCC NO: M2025735 or CCTCC NO: M2025734) are as follows: glucose 20-40 g / L, yeast extract 3-8 g / L, sodium nitrate 1-3 g / L, magnesium sulfate 0.5-1.5 g / L, dipotassium hydrogen phosphate 1-2 g / L, ferrous sulfate 0.01-0.03 g / L, potassium chloride 0.5-1.5 g / L, pH 6-7. The culture temperature is 25-35℃, the shaking speed is 120-180 rpm, and the culture time is 1-7 days.

[0081] Example 2: Synthesis of 4α-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin (Product I) using Penicillium purpureum (accession number CCTCC NO:M2025735).

[0082] Strain cultivation: Slant agar medium formula: glucose 30 g / L, magnesium sulfate 1.0 g / L, potassium dihydrogen phosphate 1.5 g / L, thiamine hydrochloride 0.1 g / L, agar 20 g / L, and water extract of potato (200 g) to a final volume of 1.0 L. Incubation temperature: 25-30℃, incubation time: 4-5 days. Primary and secondary liquid seed culture medium formula: glucose 30 g / L, yeast extract 5 g / L, sodium nitrate 2 g / L, magnesium sulfate 1.0 g / L, dipotassium hydrogen phosphate 1.5 g / L, ferrous sulfate 0.02 g / L, potassium chloride 1.0 g / L, pH 6-7. Incubation temperature: 25-30℃, shaker speed: 120-180 rpm, incubation time: 3-4 days.

[0083] Biotransformation: 4′-demethylepiotatoxin (DMEP) and 5-fluorobenzoxazole (5F-Bo) were added to the fermentation medium (pH 7.0 liquid fermentation medium containing 15 g / L carbon source, 4 g / L nitrogen source, and 1.5 g / L inorganic salts) to a final concentration of 0.3 g / L and 0.1 g / L, respectively. The culture was then inoculated with a secondary liquid seed culture of Penicillium purpureus with preservation number CCTCC NO:M2025735 at an inoculation rate of 5% (v / v) and cultured on a shaker at 25-30℃ and 120-180 rpm for 5 days.

[0084] 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%).

[0085] Figure 1 The diagram shown is a biotransformation route for the stereoselective synthesis of 4α-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin by *Penicillium purpureum* strain CCTCC NO:M2025735 in this embodiment. Figure 2 The image shows the product prepared in this embodiment. 1 H-NMR, Figure 3 For the preparation of the product 13 C-NMR, as can be seen from the figure, shows that this embodiment successfully prepared 4α-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin.

[0086] Secondary seed fermentation broth of *Penicillium purpurogenum* (accession number CCTCC NO: M2025735) was prepared using the same method described above. Secondary fermentation was carried out for 7 days. The supernatant of the fermentation broth was collected by centrifugation to obtain *Penicillium purpurogenum* fermentation product with cell removal. Biotransformation: 5% (v / v) of the supernatant of *Penicillium purpurogenum* fermentation broth with cell removal (accession number CCTCC NO: M2025735) was added to a reaction system with final concentrations of 0.3 g / L for 4′-demethylepiotatoxin (DMEP) and 0.1 g / L for 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.

[0087] Example 3: Synthesis of 4β-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin (product II) using Penicillium purpureum with accession number CCTCC NO:M2025734.

[0088] Strain culture: Slant culture, primary and secondary liquid seed culture were carried out according to the culture conditions of Example 2.

[0089] Biotransformation: 4′-demethylepiotatoxin (DMEP) and 5-fluorobenzoxazole (5F-Bo) were added to the fermentation medium at final concentrations of 0.3 g / L and 0.1 g / L, respectively. The medium was then inoculated with a secondary liquid seed culture of Penicillium purpurogenum (CCTCC NO: M2025734) at an inoculation rate of 5% (v / v). The culture was carried out at 25-30°C and 120-180 rpm on a shaker for 5 days.

[0090] Product separation and identification: The fermentation broth was treated and the product was separated and identified in the same way as in Example 2, yielding 4β-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin (product II), with a diastereomeric excess value of de96%.

[0091] Figure 4 Biotransformation route for the stereoselective synthesis of 4β-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin by Penicillium purpureum, preservation number CCTCC NO:M2025734.

[0092] Figure 5 The image shows the product prepared in this embodiment. 1 H-NMR, Figure 6 For the preparation of the product 13 C-NMR. From Figure 5 and Figure 6 The results show that 4β-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin was successfully prepared in this embodiment.

[0093] Secondary seed fermentation broth of *Penicillium purpurogenum* (accession number CCTCC NO: M2025734) was prepared using the same method described above. Secondary fermentation was carried out for 7 days. The supernatant of the fermentation broth was collected by centrifugation to obtain *Penicillium purpurogenum* fermentation product with cell removal. Biotransformation: 5% (v / v) of the supernatant of *Penicillium purpurogenum* fermentation broth with cell removal (accession number CCTCC NO: M2025734) was added to a reaction system with final concentrations of 0.3 g / L for 4′-demethylepiotatoxin (DMEP) and 0.1 g / L for 5-fluorobenzoxazole (5F-Bo). The reaction was carried out at 30°C for 3 days. The reaction product was purified and identified using the same method as in Example 2. 1 H-NMR and 13 The C-NMR spectrum shows that 4β-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin was successfully prepared.

[0094] Example 4: Detection of the antitumor activity of the product

[0095] In this embodiment, the antitumor activity of product I obtained in Example 2, namely 4α-(5″-fluorobenzoxazole)-4′-demethylepiotatoxin, was detected.

[0096] Experimental materials:

[0097] Example 2: Product I was prepared by catalysis using secondary seed liquid of Penicillium purpureum with accession number CCTCC NO: M2025735;

[0098] Control compound (etoposide, a known antitumor drug);

[0099] Tumor cell lines (HeLa, HepG2, etc.).

[0100] 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.

[0101] The test results of the antitumor activity of product I are shown in Table 1.

[0102] It can be seen that product I has a significant growth inhibitory effect on tumor cell lines such as Hela and HepG2, indicating that it has potential anti-tumor application value.

[0103] Table 1. Antitumor activity of Product I

[0104]

[0105] a IC 50 The value is the average of three repeated measurements.

[0106] 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 purpurogenum fungus, comprising the purpurogenum fungus with accession number CCTCC NO:M2025735 and / or the purpurogenum fungus with accession number CCTCC NO:M2025734.

2. A Penicillium purpureum-producing preparation, comprising the Penicillium purpureum-producing strain as described in claim 1.

3. A fermentation product of Penicillium purpureum, which is a metabolite containing bacterial cells or after removing bacterial cells, obtained by fermenting Penicillium purpureum as described in claim 1 or the bacterial preparation as described in claim 2 in a culture medium; Preferably, the Penicillium purpureum ferment is prepared according to the following method: The *Penicillium purpureum* 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 *Penicillium purpureum* fermentation product with the bacterial cells removed. Optionally, the *Penicillium purpureum* fermentation product is further dried to prepare a powder. More preferably, 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 salt; More preferably, the culture temperature is 25-45℃ and the culture time is 1-7 days.

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

5. The application according to claim 4, wherein, The Penicillium purpureus with accession number CCTCC NO:M2025735 was used to catalyze the stereoselective synthesis of 4α-(5″-fluorobenzoxazole)-4′-demethylpetropophyllotoxin from 4′-demethylpetropophyllotoxin and 5-fluorobenzoxazole.

6. The application according to claim 4, wherein, The Penicillium purpureus with accession number CCTCC NO:M2025734 was used to catalyze the stereoselective synthesis of 4β-(5″-fluorobenzoxazole)-4′-demethylpetropophyllotoxin from 4′-demethylpetropophyllotoxin and 5-fluorobenzoxazole.

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

8. A biological method for constructing thiopogonin derivatives C(sp) using Penicillium purpureum. 3 A method for stereoselectivity of α-S bonds, the method comprising: Using the *Penicillium purpureum* of claim 1, the bacterial preparation of claim 2, or the fermentation product of *Penicillium purpureum* of claim 3 as a biocatalyst, 4′-demethyleppodophyllotoxin and 5-fluorobenzoxazole are coupled via a CS bond to stereoselectively synthesize 4α-(5″-fluorobenzoxazole)-4′-demethyleppodophyllotoxin or 4β-(5″-fluorobenzoxazole)-4′-demethyleppodophyllotoxin.

9. The method according to claim 8, wherein, The reaction conditions are pH 6.0-8.0, temperature 25-35℃, reaction time 1-7 days (preferably 24-72 hours), and the molar ratio of substrate 4′-demethylepiotazotoxin to 5-fluorobenzoxazole is 1:1-1:

5.

10. The method according to claim 8, wherein, The biocatalyst used in the reaction is the Penicillium purpureum produced according to claim 1 or the bacterial preparation according to claim 2. The reaction is carried out in a culture medium, which 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 salt.

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