Microbial synthesis method of 4-cholestene-3-ketone based on cholesterol oxidase truncation

Abstract

The invention provides a method for synthesizing 4-cholestene-3-ketone based on microorganisms of a cholesterol oxidase truncation. The invention specifically provides a method for producing 4-cholestene-3-ketone, which comprises the following steps: culturing a host cell to obtain a culture product; wherein the host cell can synthesize cholesterol and heterologously express cholesterol oxidase; the cholesterol oxidase truncation is selected from cholesterol oxidase without a complete signal peptide function; and extracting and separating the target product 4-cholestene-3-ketone from the culture product of the host cell. According to the invention, the conversion from cholesterol to 4-cholestene-3-ketone can be directly completed in a living cell environment.

Description

Technical Field

[0001] This invention relates to a method for producing 4-cholesten-3-one, specifically to a method for the microbial synthesis of 4-cholesten-3-one based on a truncated form of cholesterol oxidase. Background Technology

[0002] Steroid compounds are the second largest class of drugs after antibiotics, occupying an important position in the pharmaceutical field and widely used in anti-inflammatory, endocrine therapy, anti-allergy, and anti-tumor applications. Among them, 4-cholest-4-en-3-one is an important product of cholesterol oxidase catalyzing cholesterol production. It can serve as a key raw material or intermediate for steroid drugs such as progesterone, and also possesses independent pharmacological activities, such as inhibiting fat accumulation and treating liver diseases.

[0003] Cholesterol oxidase (EC 1.1.3.6) is a flavin oxidoreductase that catalyzes the dehydrogenation of the hydroxyl group at the C3 position of cholesterol, followed by isomerization, ultimately producing 4-cholesten-3-one and hydrogen peroxide. This enzyme is typically secreted by microorganisms such as Streptomyces sp., Rhodococcus sp., and Brevibacterium sp.

[0004] Cholesterol oxidase has attracted widespread attention in biocatalytic pharmaceuticals due to its key role in converting cholesterol to 4-cholesten-3-one. The preparation of 4-cholesten-3-one from cholesterol mainly utilizes microbial fermentation to produce cholesterol oxidase, followed by extraction and separation for in vitro catalysis. CN109971748A discloses a method using a high-yield mutant strain of cholesterol oxidase obtained through mutagenesis. After fermentation, the supernatant is collected, and the cholesterol oxidase solution is obtained by centrifugation. Finally, the conversion of cholesterol to 4-cholesten-3-one is carried out in an aqueous / organic two-phase system. CN1328133A discloses a method for catalyzing the production of 4-cholesten-3-one using cholesterol oxidase secreted extracellularly by the brevicellar strain DGCDC-82. This method also utilizes the extracellular enzyme in the fermentation broth supernatant to carry out the cholesterol conversion reaction in an aqueous / organic two-phase system to obtain 4-cholesten-3-one.

[0005] However, existing technologies for producing cholesterol oxidase through microbial fermentation are highly secretion-dependent, limiting production efficiency. After microbial / heterologous expression, the protein is secreted into the fermentation broth, and its efficiency is significantly affected by the host's physiological state, intracellular folding rate, and secretion system saturation, often resulting in low expression levels, long fermentation cycles, and unstable enzyme activity. Furthermore, due to the complexity of the secretion process, pH, ionic strength, and protease activity in the extracellular environment can easily inactivate the enzyme. In addition, the fermentation broth requires centrifugation, filtration, and extraction to separate the extracellular enzyme solution before the reaction can proceed in an aqueous / organic biphase system, which is cumbersome, yield-limiting, and unsuitable for industrial scale-up. Moreover, cholesterol, due to its extremely poor water solubility, requires in vitro conversion in an aqueous / organic biphase system (typically requiring the addition of organic solvents such as n-octane or toluene / benzene / n-hexane), which not only increases the risk of environmental pollution but also limits conversion efficiency due to substrate partitioning at the two-phase interface. Summary of the Invention

[0006] This invention utilizes molecular biology techniques to truncate the gene encoding cholesterol oxidase, constructing a truncated variant of cholesterol oxidase without disrupting its correct folding and functional structure. After the truncated gene is constructed, it is transformed into host cells capable of synthesizing and accumulating cholesterol substrates, thus providing both the substrate (cholesterol) and the biocatalyst (cholesterol oxidase) within the cell, enabling the direct conversion of cholesterol to 4-cholesten-3-one in a living cellular environment. Figure 1 ).

[0007] Specifically, on one hand, the present invention provides a method for producing 4-cholesten-3-one, the method comprising:

[0008] A host cell is cultured to obtain a culture product; wherein the host cell is capable of synthesizing cholesterol and heterologously expressing cholesterol oxidase; the truncated form of cholesterol oxidase is selected from cholesterol oxidases that do not possess the complete signal peptide function;

[0009] The target product, 4-cholesten-3-one, was extracted and isolated from the culture product of the host cells.

[0010] According to some specific embodiments of the present invention, in the method of the present invention, the cholesterol oxidase is a cholesterol oxidase derived from Streptomyces or Brachybacterium.

[0011] According to some specific embodiments of the present invention, in the method of the present invention, the cholesterol oxidase is a cholesterol oxidase derived from Streptomyces sp. strain SA-COO.

[0012] According to some specific embodiments of the present invention, in the method of the present invention, the truncated cholesterol oxidase is a truncated form of wild-type cholesterol oxidase with partial or complete removal of the signal peptide sequence.

[0013] According to some specific embodiments of the present invention, in the method of the present invention, the cholesterol oxidase truncated form is selected from:

[0014] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2;

[0015] (b) A variant protein having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) based on the amino acid sequence shown in SEQ ID NO: 2 and having substantially the same catalytic activity as the protein composed of the amino acid sequence shown in SEQ ID NO: 2.

[0016] According to some specific embodiments of the present invention, in the method of the present invention, the host cell is selected from eukaryotic microorganisms. Preferably, the host cell is one or more of Saccharomyces cerevisiae or Pichia pastoris.

[0017] According to some specific embodiments of the present invention, in the method of the present invention, the host cell is Saccharomyces cerevisiae strain IMX581.

[0018] According to some specific embodiments of the present invention, in the method of the present invention, the host cells capable of synthesizing cholesterol and heterologously expressing cholesterol oxidase are prepared according to the following method:

[0019] By introducing the zebrafish DHCR7 gene and the chicken DHCR24 gene into host cells, host cells capable of synthesizing cholesterol were obtained.

[0020] A plasmid vector containing a gene encoding a truncated form of cholesterol oxidase was transferred into a cholesterol-producing host cell to obtain a host cell capable of synthesizing cholesterol and heterologously expressing cholesterol oxidase.

[0021] According to some specific embodiments of the present invention, the plasmid transfer method in the method of the present invention is the lithium acetate chemical conversion method.

[0022] According to some specific embodiments of the present invention, the process of culturing the host cells in the method of the present invention includes:

[0023] The host cells were inoculated into a growth medium for growth culture, and then transferred to a fermentation medium for induced expression or continuous fermentation to promote the expression of the cholesterol oxidase truncated form and generate 4-cholesten-3-one.

[0024] According to some specific embodiments of the present invention, in the method of the present invention, the growth medium contains 1%-5% (w / v) of available carbon source.

[0025] According to some specific embodiments of the present invention, in the method of the present invention, the fermentation medium contains 1%-5% (w / v) of induction means, wherein the induction means are selected from one or more of adding chemical inducers, changing carbon sources or changing physical culture conditions.

[0026] In some specific embodiments of the present invention, the process of culturing the host cells in the method of the present invention includes: inoculating positive transformants into shake tubes containing glucose-containing Sc-URA medium and culturing them overnight on a shaker; the next day, removing the shake tubes, centrifuging, discarding the supernatant medium, suspending the cells with sterile water, and then inoculating them into shake flasks containing galactose-containing Sc-URA medium for fermentation on a shaker.

[0027] According to some specific embodiments of the present invention, the process of extracting and isolating the target product 4-cholesten-3-one from the culture product of the host cell in the method of the present invention includes:

[0028] The target product was extracted from the fermented cells using an organic solvent and then separated by centrifugation or filtration to obtain the target product 4-cholesten-3-one.

[0029] In some specific embodiments of the present invention, the process of extracting and separating the target product 4-cholesten-3-one from the culture product of the host cell in the method of the present invention includes: adding ethyl acetate to the fermented and separated bacterial cells, taking the corresponding number of dedicated centrifuge tubes, adding glass beads, and then adding the bacterial cells to the dedicated centrifuge tubes; ultrasonically breaking the cell wall; taking the turbid liquid into an EP tube, centrifuging, and taking the supernatant through an organic filter membrane to obtain the target product 4-cholesten-3-one.

[0030] On the other hand, the present invention also provides a nucleic acid molecule that encodes a truncated form of cholesterol oxidase, wherein the truncated form of cholesterol oxidase is a wild-type cholesterol oxidase with some or all of its signal peptide sequence removed.

[0031] According to some specific embodiments of the present invention, in the technical solution of the nucleic acid molecule of the present invention, the cholesterol oxidase truncated form is as described above, specifically, it can be selected from:

[0032] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2;

[0033] (b) A variant protein having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) based on the amino acid sequence shown in SEQ ID NO: 2 and having substantially the same catalytic activity as the protein composed of the amino acid sequence shown in SEQ ID NO: 2.

[0034] According to some specific embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO: 1.

[0035] On the other hand, the present invention also provides an expression vector containing the nucleotide sequence of the nucleic acid molecule described in the present invention.

[0036] According to some specific embodiments of the present invention, the expression vector comprises a strongly inducible promoter GAL1 and a terminator CYC1.

[0037] According to some specific embodiments of the present invention, the expression vector is pESC-URA.

[0038] On the other hand, the present invention also provides a host cell capable of synthesizing cholesterol and heterologously expressing cholesterol oxidase; the truncated form of cholesterol oxidase is a truncated form of wild-type cholesterol oxidase with some or all of the signal peptide sequence removed.

[0039] According to some specific embodiments of the present invention, in the host cells of the present invention, the truncated form of cholesterol oxidase, as described above, specifically, may be selected from:

[0040] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2;

[0041] (b) A variant protein having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) based on the amino acid sequence shown in SEQ ID NO: 2 and having substantially the same catalytic activity as the protein composed of the amino acid sequence shown in SEQ ID NO: 2.

[0042] According to some specific embodiments of the present invention, the host cell is selected from eukaryotic microorganisms. Preferably, the host cell is one or more of Saccharomyces cerevisiae or Pichiapastoris.

[0043] In some specific embodiments of this invention, *Saccharomyces cerevisiae* was selected as the engineered host to express the truncated cholesterol oxidase. While *Saccharomyces cerevisiae* possesses an endoplasmic reticulum-Golgi apparatus secretion pathway, this invention utilizes the truncated enzyme to directly synthesize the enzyme intracellularly. During construction, the yeast dual-expression vector pESC-URA was used, and under the control of the GAL1 promoter, high-level expression of the target enzyme protein was induced by galactose, allowing the enzyme to be normally expressed intracellularly in a soluble form. Experimental results showed that the truncated cholesterol oxidase still possessed catalytic activity, and a characteristic peak consistent with the retention time of the 4-cholesten-3-one standard was detected in the extract of the fermentation product; furthermore, the mass spectrometry fragment ion map was consistent with the standard, confirming the formation of the target product, 4-cholesten-3-one.

[0044] Traditional processes require the addition of cholesterol substrates to the reaction system, while this invention employs metabolic engineering to enable yeast cells to synthesize and accumulate cholesterol substrates themselves. This invention genetically modifies the sterol metabolism pathway of *Saccharomyces cerevisiae*, enabling it to produce and accumulate cholesterol intracellularly, and induces the expression of the aforementioned truncated cholesterol oxidase in this modified yeast. In this way, the yeast cell simultaneously possesses both the substrate (cholesterol) and the biocatalyst (cholesterol oxidase), thus enabling the direct conversion of cholesterol to 4-cholesten-3-one in a living cellular environment. Experimental results demonstrate that, in a pure aqueous fermentation system without the addition of any organic solvents, after a period of induction culture, this invention successfully detected a large accumulation of 4-cholesten-3-one product in yeast cells (GCMS detection signal intensity reached 1 x 10⁻⁶). 7 This signifies the first successful realization of cholesterol oxidation within a living cell system, with engineered yeast cells themselves converting cholesterol into 4-cholesten-3-one. This groundbreaking result demonstrates that cholesterol oxidase-catalyzed reactions can proceed efficiently within the cellular environment, without the need for extracellular secretion or in vitro organic phase assistance as is the case in traditional methods. Attached Figure Description

[0045] Figure 1 This is a schematic diagram of the biosynthetic pathway of 4-cholesten-3-one.

[0046] Figure 2 This is a map of cholesterol oxidase expression plasmids.

[0047] Figure 3 The results demonstrate the verification of the catalytic activity of truncated cholesterol oxidase in cells.

[0048] Figure 4 This shows the fermentation yield of 4-cholesten-3-one by the Saccharomyces cerevisiae strain. Detailed Implementation

[0049] The embodiments of the present invention are further described below with reference to specific examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to be limiting. Therefore, the present invention should not be construed as limited to the following embodiments, but should be construed as covering any and all variations that become apparent from the teachings provided herein.

[0050] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0051] Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions in the art or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0052] Unless otherwise specified, all materials and reagents used in the examples are commercially available products.

[0053] Example 1: Production of 4-cholesten-3-one from cholesterol synthesized intracellularly by a truncated form of cholesterol oxidase.

[0054] 1. Design and acquisition of the gene sequence of the truncated cholesterol oxidase.

[0055] The cholesterol oxidase selected in this invention is derived from Streptomyces sp. (strain SA-COO) strain, and its encoding gene is choA (GenBank sequence number: M31939.1).

[0056] This invention designs a truncated form of cholesterol oxidase, namely the truncated cholesterol oxidase (its amino acid sequence is shown in SEQ ID NO: 2), and optimizes the chassis codon of the wild-type cholesterol oxidase choA gene in Saccharomyces cerevisiae to obtain the gene tchoA encoding the truncated cholesterol oxidase, whose nucleotide sequence is shown in SEQ ID NO: 1.

[0057] The two DNA fragments, choA and tchoA, were synthesized by Nanjing Genscript Biotech Co., Ltd.

[0058] SEQ ID NO:1:

[0059]

[0060] SEQ ID NO:2:

[0061] MDNGGYVPAVVIGTGYGAAVSALRLGEAGVQTLMLEMGQLWNQPGPDGNIFCGMLNPDKRSSWFKNRTEAPLGSFLWLDVVNRNIDPYAGVLDRVNYDQMSVYVGRGVGGGSLVNGGMAVEPKRSY FEEILPRVDSSEMYDRYFPRANSMLRVNHIDTKWFEDTEWYKFARVSREQAGKAGLGTVFVPNVYDFGYMQREAAGEVPKSALATEVIYGNNHGKQSLDKTYLAAALGTGKVTIQTLHQVKTIRQT KDGGYALTVEQKDTDGKLLATKEISCRYLFLGGSLGSTELLVRARDTGTLPNLNSEVGAGWGPNGNIMTARANHMWNPTGAHQSSIPALGIDAWDNSDSSVFAEIAPMPAGLETWVSLYLAITKN PQRGTFVYDAATDRAKLNWTRDQNAPAVNAAKALFDRINKANGTIYRYDLFGTQLKAFADDFCYHPLGGCVLGKATDDYGRVAGYKNLYVTDGSLIPGSVGVNPFVTITALAERNVERIIKQDVTAS

[0062] 2. Construction of recombinant expression vectors

[0063] The *Saccharomyces cerevisiae* expression vector pESC-URA (Novopro, catalog number #V010319) was selected. This vector contains the strongly inducible promoter GAL1 and terminator CYC1, making it suitable for protein expression in yeast. The construction process is as follows: First, the pESC-URA plasmid was linearized by double digestion with restriction endonucleases BamHI-HF (NEB, catalog number #R3136S) and HindIII-HF (NEB, catalog number #R3136S), and the large fragment was recovered. Next, the target gene fragment (choA or tchoA) was directionally cloned into the linearized vector using the ClonExpress II One Step Cloning Kit (Vazyme, catalog number #C112-01).

[0064] The ligation product was added to 100 μL of Top10 *E. coli* chemicompetent cells (Kangti Life, catalog number #KL101), gently mixed, and incubated on ice for 30 minutes. Subsequently, the cells were heat-shocked at 42°C for 90 seconds and immediately cooled on ice for 2 minutes. 700 μL of LB broth was added, and the cells were incubated at 37°C and 200 rpm for 1 hour. The cells were collected by centrifugation, and 100 μL of the suspension was spread onto LB agar plates containing 100 μL of ampicillin and incubated overnight at 37°C.

[0065] The following day, well-grown single colonies were selected and colony PCR was performed using primers G10-F (5'-GGTGGTAATGCCATGTAATATG-3', SEQ ID NO: 3) and G10-R (5'-TTCTGGCAAGGTAGACAAG-3', SEQ ID NO: 4) for verification. The colony PCR reaction was performed using 2× Phanta Max Master Mix (Vazyme, catalog number #P525-01). The PCR program was as follows: pre-denaturation at 95°C for 3 minutes; followed by 30 cycles: denaturation at 95°C for 15 seconds, annealing at 55°C for 15 seconds, extension at 72°C for 30 seconds; and finally, a complete extension at 72°C for 5 minutes. After amplification, 5 μL of the PCR product was examined by 1% agarose gel electrophoresis to verify band size. The samples were then sent to Sanger sequencing at Sangon Biotech Co., Ltd. Sequencing results, through sequence alignment analysis, confirmed the acquisition of recombinant expression plasmids pESC-URA-choA and pESC-URA-tchoA with completely correct sequences. Figure 2 ).

[0066] 3. Construction of functional strains for heterologous expression of cholesterol oxidase to produce 4-cholesten-3-one

[0067] The original chassis used in this invention is the Saccharomyces cerevisiae strain IMX581, with the genotype MATa ura3-52 can1Δ::cas9-natNT2 TRP1 LEU2 HIS3. This strain is a standardized chassis that is publicly available and widely used in academia. For its specific construction method and characteristics, please refer to: Mans R, et al., FEMS Yeast Research, 2015, 15(4). First, referring to the method of Cheng J et al. (Plant Communications, 2020, 2), the zebrafish DHCR7 gene and the chicken DHCR24 gene (synthesized by Nanjing GenScript) were introduced into the IMX581 strain. Specifically, the Cas9 protein integrated and expressed by IMX581 was combined with guide RNA (gRNA) plasmids targeting the ERG5 and ERG6 sites, and the expression frames of the two genes were integrated into the genome through homologous recombination. GC-MS detection confirmed that it could synthesize cholesterol, and the strain was named IMX581-CHO.

[0068] To achieve the conversion of endogenous cholesterol into the target product, this experiment involved transforming a recombinant plasmid carrying the cholesterol oxidase encoding gene into a chassis strain. The constructed expression plasmids pESC-URA-choA and pESC-URA-tchoA were transformed into the chassis strain IMX581-CHO using the LiAc / SS carrier DNA / PEG transformation method (referencing Gietz RD, et al. Nat Protoc. 2007). Transformation steps: IMX581-CHO bacterial culture in the logarithmic growth phase (OD=0.6-0.8) was collected by centrifugation, and the cells were washed and resuspended sequentially with sterile water and 0.1 M lithium acetate (LiAc) solution. In 50 μL of competent cells, 50 μg SS-DNA, 1 μg target plasmid DNA, and 240 μL 50% (w / v) PEG3350 were added sequentially, and water was added to a final volume of 360 μL. After mixing, incubate at 30°C for 30 minutes, followed by heat shock at 42°C for 20 minutes. Centrifuge to recover the bacterial cells, resuspend, and plate on SD-URA deficient medium (formulation: 2% glucose, 0.67% YNB, 0.077% CSM-Ura, 2% agar). After incubation at 30°C for 3-5 days, the plasmid was successfully introduced by colony PCR as described above, obtaining recombinant engineered strains IMX581-CHO+pESC-URA-choA and IMX581-CHO+pESC-URA-tchoA.

[0069] 4. Production of 4-cholesten-3-one by shake-flask fermentation

[0070] The verified bacterial strain was inoculated into 3 mL of SD-URA liquid medium (SC-ura Broth, Solarbio, supplemented with 2% glucose) and cultured overnight at 30°C and 220 rpm for 16-18 hours. The cells were collected by centrifugation at 4000 rpm for 5-10 min, the supernatant was discarded in a clean bench, and the cells were resuspended in 1 mL of sterile water. The cells were then inoculated into 50 mL of induction fermentation medium (SC-ura Broth, Solarbio, supplemented with 2% galactose) at a 2% inoculation rate. The shake flasks were placed in a shaker at 30°C and 220 rpm for 72 hours for continuous fermentation. After fermentation, the cells were collected by centrifugation. 2 mL of ethyl acetate was added to the cell precipitate as an extraction solvent. The cells were then homogenized using a sample homogenizer for 30 min to disrupt the cell wall. Subsequently, the mixture was centrifuged at 13,000 rpm for 5 min, and the ethyl acetate organic phase containing the target product was aspirated, filtered through a 0.22 μm organic filter membrane, and then analyzed by GC-MS.

[0071] Detection: Analysis was performed using an Agilent 8890-7000D gas chromatograph-mass spectrometer equipped with an HP-5msUI (30 m × 0.25 mm × 0.25 μm) ultra-inert capillary column. Specific detection parameters were as follows: Injector: 300°C, splitless injection, injection volume 1 μL; Temperature program: Initial temperature 240°C, hold for 5 min; then ramp to 300°C at a rate of 10°C / min and hold at 300°C for 25 min; Mass spectrometry conditions: Electron impact source (EI); Ion source temperature 230°C; Quadrupole temperature 150°C; Full scan mode, scan range m / z 50–550.

[0072] See the results of gas chromatography-mass spectrometry (GC-MS) for the detection of yeast extract. Figure 3 In this embodiment, the yeast extract has the same retention time as the target product 4-cholesten-3-one. This demonstrates that the truncated cholesterol oxidase of the present invention possesses catalytic activity within cells.

[0073] The yield of 4-cholesten-3-one produced by the Saccharomyces cerevisiae strain in this embodiment is shown in the figure. Figure 4The results showed that only a small amount of 4-cholesten-3-one was detected in the cells of the control strain (expressing wild-type cholesterol oxidase), while a large amount of 4-cholesten-3-one was detected in the cells of the experimental strain of the present invention (expressing a truncated form of cholesterol oxidase), with a yield 50 times that of the control group, indicating a significant transformation effect.

[0074] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A method for producing 4-cholesten-3-one, the method comprising: A host cell is cultured to obtain a culture product; wherein the host cell is capable of synthesizing cholesterol and heterologously expressing cholesterol oxidase; the truncated form of cholesterol oxidase is selected from cholesterol oxidases that do not possess the complete signal peptide function; The target product, 4-cholesten-3-one, was extracted and isolated from the culture product of the host cells.

2. The method according to claim 1, wherein, The cholesterol oxidase is a cholesterol oxidase derived from Streptomyces or Brachybacterium. Preferably, the cholesterol oxidase is a cholesterol oxidase derived from Streptomyces sp. strain SA-COO; Preferably, the truncated form of cholesterol oxidase is a truncated form of wild-type cholesterol oxidase with some or all of the signal peptide sequence removed; Preferably, the cholesterol oxidase truncated form is selected from: (a) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2; (b) A variant protein having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) based on the amino acid sequence shown in SEQ ID NO: 2 and having substantially the same catalytic activity as the protein composed of the amino acid sequence shown in SEQ ID NO:

2.

3. The method according to claim 1 or 2, wherein, The host cells are selected from eukaryotic microorganisms; Preferably, the host cell is one or more of Saccharomyces cerevisiae or Pichia pastoris; Preferably, the host cell is Saccharomyces cerevisiae strain IMX581.

4. The method according to any one of claims 1-3, wherein, Host cells capable of synthesizing cholesterol and heterologously expressing cholesterol oxidase were prepared according to the following method: By introducing the zebrafish DHCR7 gene and the chicken DHCR24 gene into host cells, host cells capable of synthesizing cholesterol were obtained. A plasmid vector containing a gene encoding a truncated form of cholesterol oxidase was transferred into a cholesterol-producing host cell to obtain a host cell capable of synthesizing cholesterol and heterologously expressing cholesterol oxidase.

5. The method according to any one of claims 1-4, wherein, The process of culturing the host cells includes: The host cells were inoculated into a growth medium for growth culture, and then transferred to a fermentation medium for induced expression or continuous fermentation to promote the expression of the cholesterol oxidase truncated form and generate 4-cholesten-3-one.

6. The method according to claim 5, wherein, The growth medium contains 1%-5% (w / v) of available carbon source; Preferably, the fermentation medium contains 1%-5% (w / v) of induction methods, wherein the induction methods are selected from one or more of adding chemical inducers, changing the carbon source, or changing the physical culture conditions.

7. The method according to any one of claims 1-6, wherein, The process of extracting and isolating the target product 4-cholesten-3-one from the culture product of the host cells includes: The target product was extracted from the fermented cells using an organic solvent and then separated by centrifugation or filtration to obtain the target product 4-cholesten-3-one.

8. A nucleic acid molecule encoding a truncated form of cholesterol oxidase, wherein the truncated form of cholesterol oxidase is a wild-type cholesterol oxidase with partial or complete removal of the signal peptide sequence; Preferably, the cholesterol oxidase truncated form is selected from: (a) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2; (b) A variant protein having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) based on the amino acid sequence shown in SEQ ID NO: 2 and having substantially the same catalytic activity as the protein composed of the amino acid sequence shown in SEQ ID NO: 2; Preferably, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO:

1.

9. An expression vector containing the nucleotide sequence of the nucleic acid molecule of claim 8; Preferably, the expression vector comprises a strongly inducible promoter GAL1 and a terminator CYC1; Preferably, the expression vector is pESC-URA.

10. A host cell capable of synthesizing cholesterol and heterologously expressing cholesterol oxidase; wherein the truncated form of cholesterol oxidase is a truncated form of wild-type cholesterol oxidase with partial or complete removal of the signal peptide sequence; Preferably, the cholesterol oxidase truncated form is selected from: (a) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2; (b) A variant protein having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) based on the amino acid sequence shown in SEQ ID NO: 2 and having substantially the same catalytic activity as the protein composed of the amino acid sequence shown in SEQ ID NO: 2; Preferably, the host cell is selected from eukaryotic microorganisms; more preferably, the host cell is one or more of Saccharomyces cerevisiae or Pichia pastoris. Preferably, the host cell is Saccharomyces cerevisiae strain IMX581.

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