Microorganism for producing sterol derivative and use thereof

Abstract

Provided are a microorganism for producing a sterol derivative and a use thereof.

Description

Microorganisms that produce sterol derivatives and their uses Technical Field

[0001] This invention belongs to the field of industrial microbiology, specifically relating to microorganisms that produce sterol derivatives and their uses. Background Technology

[0002] Sterols are a class of steroids, specifically cyclopentane-polyhydrophenanthrene derivatives composed of three hexane rings and one cyclopentane ring, characterized by the presence of a hydroxyl group at the carbon-3 position. Except in bacteria, sterols are widely found in the cells and tissues of animals and plants. Sterols have various biological functions, such as serving as components of cell membranes and constituting adrenal cortex hormones and sex hormones. In animals, cholesterol is the most abundant sterol, and it can be converted into steroid hormones in the body, including progesterone, estradiol, testosterone, cortisol, and aldosterone, playing crucial roles in the human body.

[0003] Due to the important role of steroids in medicine, developing cost-effective production methods has been a research hotspot. Phytosterols are structurally similar to animal sterols and are widely found in plants, making extraction relatively easy, while the extraction and acquisition costs of animal sterols are high. Therefore, phytosterols are suitable as economical raw materials for the industrial production of steroid drugs or pharmaceutical intermediates. Currently, steroid production typically begins with the bioconversion of phytosterols into key starting materials via microorganisms, followed by further chemical, enzymatic, or biological steps to generate steroid intermediates and the final drug.

[0004] Some microorganisms possess the ability to metabolize sterols as their carbon and energy source. Among them, mycobacteria are widely used in the industrial production of steroids. They can convert 5-en-3-ol sterol substrates into 4-en-3-one products through sterol side chain modification and sterol ring modification (Figure 1). However, the 5-en-3-ol steroid products require further processing through in vitro chemical methods or chemoenzymatic methods. For example, dehydroepiandrosterone (DHEA) is an over-the-counter supplement and an important intermediate for steroidal active pharmaceutical ingredients. A common preparation method involves first using microorganisms to bioconvert phytosterols into 4-androstene-3,17-dione (AD), followed by chemical or chemoenzymatic synthesis to obtain DHEA (Fryszkowska, A. et al., Development of a Chemoenzymatic Process for Dehydroepiandrosterone Acetate Synthesis. Org. Process Res. Dev. 20, 1520–1528 (2016); Su, B.-M. et al. A chemoenzymatic process for preparation of highly purified dehydroepiandrosterone in high space-time yield. Bioorganic Chem. 133, 106391 (2023); Zhou et al., Efficient Biotransformation of Phytosterols to Dehydroepiandrosterone by Mycobacterium sp. Appl. Biochem. Biotechnol. 186, 496-506 (2018)). Another published method for preparing 5-en-3-ol sterol derivatives using phytosterols as substrates via a biological process requires, to avoid oxidation of the 3-hydroxyl group of the sterol ring, first reacting the 3-hydroxyl group of the phytosterol ring with dimethoxymethane using phosphorus pentoxide as a catalyst to generate 3-methoxymethylated phytosterol. This is then converted into 3-methoxymethylated sterol derivatives, such as 3-methoxymethylated DHEA or pregnenolone, through microbial fermentation.Then, DHEA and progesterone were obtained by acid hydrolysis (Zhou et al., Efficient biotransformation of phytosterols to dehydroepiandrosterone by Mycobacterium sp. Appl. Biochem. Biotechnol. 186, 496-506. (2018); Karpov et al., Pregnenolone and progesterone production from natural sterols using recombinant strain of Mycolicibacterium smegmatis mc2155 expressing mammalian steroidogenesis system. Microb. Cell Fact. 23, 105. (2024)).

[0005] While the above-mentioned schemes all use biological methods in some processes, they still involve complex chemical reactions and require the use of irritating solvents and hazardous chemicals. This not only increases manufacturing and waste disposal costs but also causes environmental problems.

[0006] Therefore, it is necessary to develop more economical, efficient, and environmentally friendly sterol derivative production processes to reduce the use of chemicals and solvents and directly convert phytosterols into sterol derivatives in the form of 5-en-3-ol through biotransformation, which would be beneficial for the production of steroid drugs or pharmaceutical intermediates. Summary of the Invention

[0007] Many microorganisms can degrade sterols; for example, some microorganisms can convert cholesterol or phytosterols into steroidal anti-inflammatory drugs (AD) or their derivatives. The metabolic pathway for converting sterols to AD involves the complete degradation of the sterol side chain and the isomerization of the sterol ring's CH-OH at position 3 to C=O and the double bond at position 5 to position 4. Two enzymes have been reported to catalyze the reaction of converting the sterol ring's CH-OH at position 3 to C=O and the double bond at position 5 to position 4: cholesterol oxidase and 3β-hydroxysteroid dehydrogenase / isomerase (Kreit, Microbial catabolism of sterols: focus on the enzymes that transform the sterol 3β-hydroxy-5-en into 3-keto-4-en. FEMS Microbiol Lett. 364(3)(2017)).

[0008] In microorganisms capable of converting sterols to steroids (AD), the production of AD can be blocked and DHEA produced if proteins catalyzing sterol epoxidation and isomerization can be found and disrupted (Figure 2). Similarly, microorganisms with disrupted proteins can also be used to produce other 5-en-3-ol sterol derivatives, such as pregnenolone, 17α-hydroxypregnenolone, 7-dehydrocholesterol, 25-hydroxycholesterol, 22(S)-hydroxycholesterol, 7β-hydroxycholesterol, 20α-hydroxycholesterol, 27-hydroxycholesterol, and androstenediol. For example, WO2024131933A1 discloses an engineered mycobacterium capable of converting phytosterols into progesterone in a one-step fermentation process. Based on this engineered mycobacterium, by disrupting the proteins that catalyze sterol epoxidation and isomerization reactions, an engineered mycobacterium capable of converting phytosterols into pregnenolone in a one-step process can be obtained.

[0009] This invention discovers that the redox cofactor mycofactocin (MFT) or MFT-dependent oxidoreductases play a crucial role in the oxidation of sterol rings. By inhibiting the expression or function of MFT-related proteins, particularly by inhibiting the expression or function of cholesterol oxidase and / or 3β-hydroxysteroid dehydrogenase / isomerase, the oxidation of sterol rings can be effectively suppressed, thereby facilitating the production of sterol derivatives in the form of 5-en-3-ols, such as DHEA, pregnenolone, 7-dehydrocholesterol, 17-hydroxypregnenolone, 25-hydroxycholesterol, 22(S)-hydroxycholesterol, 7β-hydroxycholesterol, 20α-hydroxycholesterol, 27-hydroxycholesterol, and androstenediol.

[0010] Accordingly, in a first aspect, the present invention provides an engineered microorganism in which the expression and / or activity of cholesterol oxidase and / or 3β-hydroxysteroid dehydrogenase / isomerase are inhibited, and the expression and / or activity of one or more redox cofactor mycofactocin (MFT)-related proteins are inhibited.

[0011] In some embodiments, the engineered microorganisms of the present invention are derived from the genera *Mycobacterium*, *Streptomyces*, *Geodermatophilus*, *Nocardiodes*, *Frankia*, *Pseudonocardia*, *Gordonia*, *Nocardia*, or *Rhodococcus*. In some preferred embodiments, the engineered microorganisms of the present invention are derived from the genus *Mycobacterium*. In some more preferred embodiments, the engineered microorganisms of the present invention are derived from *Mycobacterium tuberculosis*, *Mycobacterium pseudoocculta*, *Mycobacterium occulta*, *Mycobacterium bovis*, *Mycobacterium flavum*, *Mycobacterium smegmatis*, or *Mycobacterium aureum*. In a further preferred embodiment, the engineered microorganisms of the present invention are derived from *Mycobacterium aureum*. In an even more preferred embodiment, the engineered microorganisms of the present invention are derived from the *Mycobacterium aureum* strain NRRL B-3805.

[0012] In embodiments of the present invention, cholesterol oxidase can catalyze the conversion of the CH-OH at the 3-position of the sterol ring to the C=O and the double bond at the 5-position isomerized to the 4-position. In some embodiments, the cholesterol oxidase is derived from the IPR052542 family (Cholesterol Oxidase Enzyme (IPR052542) - InterPro entry - InterPro).

[0013] In some embodiments, the cholesterol oxidase has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:20, 22-27. In some preferred embodiments, the cholesterol oxidase has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:20 or 22. In some more preferred embodiments, the cholesterol oxidase has the amino acid sequence shown in SEQ ID NO:20 or 22.

[0014] In embodiments of the present invention, the 3β-hydroxysteroid dehydrogenase / isomerase catalyzes the conversion of the CH-OH at the 3-position of the sterol ring to the C=O and the isomerization of the double bond at the 5-position to the 4-position. In some preferred embodiments, the 3β-hydroxysteroid dehydrogenase / isomerase is derived from the IPR002225 domain family (3-beta hydroxysteroid dehydrogenase / isomerase(IPR002225)-InterPro entry-InterPro).

[0015] In some embodiments, the 3β-hydroxysteroid dehydrogenase / isomerase has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:18. In some preferred embodiments, the 3β-hydroxysteroid dehydrogenase / isomerase has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:18. In some more preferred embodiments, the 3β-hydroxysteroid dehydrogenase / isomerase has the amino acid sequence shown in SEQ ID NO:18.

[0016] In embodiments of the present invention, the MFT-related protein is selected from proteins encoded by the MFT synthesis gene cluster, MFT transmethylases, and MFT-dependent oxidoreductases.

[0017] In some implementations, the protein encoded by the MFT synthesis gene cluster is selected from: proteins derived from the IPR023988 family (Mycofactocin precursor peptide (IPR023988)-InterPro entry-InterPro), proteins derived from the IPR023850 family (Peptide chaperone MftB (IPR023850)-InterPro entry-InterPro), proteins derived from the IPR023913 family (Mycofactocin maturase MftC IPR023913-InterPro entry-InterPro), proteins derived from the IPR023989 family (Pre-mycofactocin synthase (IPR023989)-InterPro entry-InterPro), proteins derived from the IPR023871 family (Mycofactocin precursor peptide peptidase (IPR023871)-InterPro entry-InterPro), proteins derived from the IPR023981 family (Pre-mycofactocin... The protein is a glycosyltransferase (IPR023981)-InterPro entry-InterPro, an oxidoreductase encoded by the MFT synthesis gene cluster, and a protein derived from the IPR023851 family (Transcriptional regulator, TetR-type IPR023851-InterPro entry-InterPro). In some preferred embodiments, the protein encoded by the MFT synthesis gene cluster is selected from: MftA, MftB, MftC, MftD, MftE, MftF, MftG, MftR, and MyAD_06490.

[0018] In some embodiments, the MFT transmethylase is derived from the NF041255 family (mycofactocin oligosaccharide methyltransferase MftM NF041255-InterPro entry-InterPro). In a preferred embodiment, the MFT transmethylase has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:78. In some more preferred embodiments, the MFT transmethylase has the amino acid sequence shown in SEQ ID NO:78.

[0019] In some embodiments, the MFT-dependent oxidoreductase is derived from the PF00106, PF00107, or PF00465 family. In some embodiments, the MFT-dependent oxidoreductase is derived from the PF00106 family.

[0020] In some preferred embodiments, the MFT-related protein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 76, 77, 78, and 79. In some more preferred embodiments, the MFT-related protein has the amino acid sequence shown in any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 76, 77, 78, and 79.

[0021] In some specific embodiments, the expression and / or activity of one or more of the following groups of proteins in the engineered microorganisms of the present invention are inhibited:

[0022] (1) 3β-hydroxysteroid dehydrogenase / isomerase and MFT-dependent oxidoreductase;

[0023] (2) 3β-hydroxysteroid dehydrogenase / isomerase and MftA;

[0024] (3) 3β-hydroxysteroid dehydrogenase / isomerase and MftB;

[0025] (4) 3β-hydroxysteroid dehydrogenase / isomerase and MftC;

[0026] (5) 3β-hydroxysteroid dehydrogenase / isomerase and MftD;

[0027] (6) 3β-hydroxysteroid dehydrogenase / isomerase and MftE;

[0028] (7) 3β-hydroxysteroid dehydrogenase / isomerase and MftF;

[0029] (8) Oxidoreductases encoded by 3β-hydroxysteroid dehydrogenase / isomerase and MFT synthesis gene cluster;

[0030] (9) 3β-hydroxysteroid dehydrogenase / isomerase, oxidoreductase and cholesterol oxidase encoded by the MFT synthesis gene cluster;

[0031] (10) 3β-hydroxysteroid dehydrogenase / isomerase, oxidoreductase encoded by MFT synthesis gene cluster, cholesterol oxidase, MftD and MFT-dependent oxidoreductase.

[0032] In some specific embodiments, the expression and / or activity of one or more of the following groups of proteins in the engineered microorganisms of the present invention are inhibited:

[0033] (1) A protein or a variant thereof having an amino acid sequence as shown in SEQ ID NO:18 and a protein or a variant thereof having an amino acid sequence as shown in SEQ ID NO:2;

[0034] (2) Proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:18 and proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:4;

[0035] (3) Proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:18 and proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:6;

[0036] (4) Proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:18 and proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:8;

[0037] (5) Proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:18 and proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:10;

[0038] (6) Proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:18 and proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:12;

[0039] (7) Proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:18 and proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:14;

[0040] (8) Proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:18 and proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:16;

[0041] (9) Proteins or variants thereof having an amino acid sequence as shown in SEQ ID NO:18, proteins or variants thereof having an amino acid sequence as shown in SEQ ID NO:16, proteins or variants thereof having an amino acid sequence as shown in SEQ ID NO:22 and proteins or variants thereof having an amino acid sequence as shown in SEQ ID NO:20;

[0042] (10) A protein or a variant thereof having an amino acid sequence as shown in SEQ ID NO:18, a protein or a variant thereof having an amino acid sequence as shown in SEQ ID NO:16, a protein or a variant thereof having an amino acid sequence as shown in SEQ ID NO:22, a protein or a variant thereof having an amino acid sequence as shown in SEQ ID NO:20, a protein or a variant thereof having an amino acid sequence as shown in SEQ ID NO:10 and a protein or a variant thereof having an amino acid sequence as shown in SEQ ID NO:2.

[0043] In some embodiments of the present invention, the expression and / or activity of the protein in the engineered microorganism is inhibited by any one or a combination of the following methods:

[0044] (1) Destroy the protein encoding gene in the engineered microorganism;

[0045] (2) Replace the promoter of the protein-encoding gene in the engineered microorganism with a weak promoter;

[0046] (3) Introduce into the engineered microorganism a molecule that inhibits the expression and / or activity of the protein.

[0047] In some preferred embodiments, the expression and / or activity of the protein in the engineered microorganism is inhibited by disrupting the gene encoding the protein in the engineered microorganism. In a further preferred embodiment, the gene encoding the protein is disrupted by homologous recombination.

[0048] The engineered microorganisms of this invention are capable of synthesizing sterol derivatives in the form of 5-en-3-ol from sterols.

[0049] In some preferred embodiments, the sterol is selected from β-sitosterol, campesterol, stigmasterol, brassosterol, β-sitosterol, campesterol and cholesterol.

[0050] In some preferred embodiments, the sterol derivative is selected from dehydroepiandrosterone (DHEA), pregnenolone, 17-hydroxypregnenolone, 7-dehydrocholesterol, androstenediol, 25-hydroxycholesterol, 22(S)-hydroxycholesterol, 7β-hydroxycholesterol, 20α-hydroxycholesterol, and 27-hydroxycholesterol.

[0051] In a second aspect, the present invention provides a method for preparing sterol derivatives, the method comprising:

[0052] (1) The engineered microorganisms described in the first aspect of the invention are cultured in the presence of sterols and under conditions suitable for the production of sterol derivatives; and / or

[0053] (2) Isolate the sterol derivative from the culture of the engineered microorganism.

[0054] In some implementations, the sterol is β-sitosterol, and the sterol derivative is DHEA.

[0055] In a third aspect, the present invention provides the use of engineered microorganisms as described in the first aspect of the present invention in the preparation of sterol derivatives.

[0056] In some implementations, the sterol is β-sitosterol, and the sterol derivative is DHEA. Attached Figure Description

[0057] Figure 1 shows the general structural formulas of sterols in the form of 5-en-3-ol (A) and sterol derivatives in the form of 4-en-3-one (B), and the chemical structural formulas of several sterol derivatives. The nomenclature (AD) of the sterol ring and the sequence of carbon atoms are shown in the figure. A chemical group is attached to the 17th carbon atom; this chemical group can be any number of carbon atoms (including 0), and can be straight-chain or branched. Chemical groups may also be attached to other carbon atoms. C. Dehydroepiandrosterone (DHEA); D. 7-Dehydrocholesterol; E. Androstenediol; F. Pregnenolone.

[0058] Figure 2 illustrates a design for producing dehydroepiandrosterone (DHEA) using Mycobacterium species capable of bioconverting phytosterols (such as β-sitosterol) to 4-androstenedione (AD). Disrupting the activity of enzymes involved in the epoxidation of sterols (AB) would redirect the bioprocess from AD production to DHEA production.

[0059] Figure 3 shows a representative map of integrative plasmids used to disrupt MyAD_21005. The only difference between the integrative plasmids used to disrupt other genes and the representative plasmid is the upstream and downstream homologous sequences.

[0060] Figure 4 shows the yields of AD and DHEA in a 96-well plate incubation experiment. The yields were determined by gas chromatography after incubating the genetically modified B-3805-derived Mycobacterium strain culture with 2 mg / well of 70% β-sitosterol for three days. Each data point (circle) represents a single replicate. The horizontal line represents the mean.

[0061] Figure 5 shows the concentrations of AD and DHEA determined in a 96-well plate incubation experiment. Cultures of the genetically disrupted Mycobacterium B-3805 strain were incubated with 2 mg / well of 70% β-sitosterol for three days and then analyzed by LC-MS. Each data point (circle) represents a single replicate. The horizontal line represents the mean of three replicates.

[0062] Figure 6 shows the LC-MS multiple reaction monitoring (MRM) chromatograms of DHEA (271.2→253.2) and AD (287.2→97.0) in a 96-well plate incubation experiment. The assay was performed after incubating the genetically modified B-3805 Mycobacterium strain culture with 2 mg / well of 70% β-sitosterol for three days. Due to the difference in ionization efficiency between DHEA and AD, their MRM chromatograms were normalized to the sample with the highest intensity for each.

[0063] Figure 7 shows a matrix of sequence identity percentages between the protein encoded by the disrupted gene disclosed in this invention and known cholesterol oxidases or 3β-hydroxysteroid dehydrogenases / isomerases in the literature. In this matrix, Mtu represents Mycobacterium tuberculosis, Msmeg represents Mycolicibacterium smegmatis, and Mneo represents Mycolicibacterium neoaurum. Detailed Implementation

[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0065] To make this disclosure easier to understand, some terms are defined below.

[0066] As used in this specification and the appended claims, the singular forms “a” and “the” include plural references, unless the context clearly specifies otherwise.

[0067] In this document, the terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., meaning “including but not limited to”).

[0068] In this document, “and / or” means and includes any and all possible combinations of one or more of the associated listed items. For example, “the composition contains A and / or B” can be interpreted as the composition contains A, the composition contains B, or the composition contains both A and B.

[0069] All numerical names, such as pH, temperature, time, concentration, and molecular weight, including ranges, are approximate values ​​that are appropriately varied in increments of 1.0 or 0.1, or optionally varied (+) or (-) by changes of + / - 15%, 10%, 5%, or 2%. It should be understood that all numerical names are preceded by the term "about". It should also be understood that the reagents described herein are exemplary only, and their equivalents are known in the art. When referring to measurable values ​​such as amount or concentration, the term "about" as used herein means a variation within 20%, 10%, 5%, 1%, 0.5%, or 0.1% of the specified amount.

[0070] In this document, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid sequence,” “nucleotide sequence,” and “polynucleotide” are used interchangeably and refer to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. Therefore, the term includes, but is not limited to, single-stranded or double-stranded DNA or RNA, genomic DNA, cDNA, DNA / RNA hybrids, or polymers comprising, consisting of, or substantially consisting of purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derived nucleotide bases.

[0071] The terms “protein,” “peptide,” “polypeptide,” and “amino acid sequence” are used interchangeably and, in their broadest sense, refer to a polymer of two or more amino acid subunits, amino acid analogs, or peptide mimics. “Protein,” “peptide,” “polypeptide,” and “amino acid sequence” contain at least two amino acids, and there is no limit to the maximum number of amino acids. As used herein, the term “amino acid” refers to natural and / or non-natural or synthetic amino acids, including D and L optical isomers and amino acid analogs.

[0072] Equivalents having one or more amino acid modifications compared to the protein or amino acid sequence described herein are also covered within the scope of this invention, provided that such modification does not affect or substantially does not affect the activity of the protein or amino acid sequence. In this document, amino acid modification can be amino acid substitution, amino acid deletion, or amino acid insertion. Amino acid substitution can be conserved or non-conserved. A conserved substitution (also called a conserved mutation, conserved replacement, or conserved variation) is an amino acid substitution in a protein that changes a given amino acid to a different amino acid having similar biochemical properties (e.g., charge, hydrophobicity, or size). In this document, "conserved substitution" means that an amino acid residue is replaced by another biologically similar residue. Examples of conserved substitution include one hydrophobic residue such as isoleucine, valine, leucine, or methionine replacing another; or one charged or polar residue replacing another, such as arginine replacing lysine, glutamic acid replacing aspartic acid, glutamine replacing asparagine, etc. Other exemplary examples of conservative substitutions include the following changes: alanine to serine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glycine to proline; histidine to asparagine or glutamine; lysine to arginine, glutamine, or glutamic acid; phenylalanine to tyrosine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and so on.

[0073] In this article, "expression" refers to the process by which a nucleic acid sequence is transcribed into mRNA and / or the transcribed mRNA is subsequently translated into peptides, polypeptides, amino acid sequences, or proteins. If the nucleic acid sequence originates from genomic DNA, expression may include the splicing of mRNA in eukaryotic cells.

[0074] When the term "coding" is applied to a nucleic acid sequence, it refers to a nucleic acid sequence that, if in its natural state or when manipulated by methods well known to those skilled in the art, can be transcribed to produce mRNA and / or translated to produce a polypeptide, is called a "coding" polypeptide sequence. Therefore, a "coding sequence" is a nucleic acid sequence that has the aforementioned function, and it can be DNA or RNA (e.g., mRNA).

[0075] In this paper, "coding gene" includes "coding sequence" and may optionally include other nucleotide sequence elements for regulating gene expression, such as promoters and terminators. Within a "coding gene," the coding sequence and other nucleotide sequence elements are arranged in a suitable order to promote the correct expression of the encoded protein.

[0076] "Disruption" of a coding gene includes disruption of the coding sequence and / or other nucleotide sequence elements (e.g., promoters, terminators, etc.), such as deleting part or all of the nucleotide sequence, inserting new nucleotide sequences, or replacing part of the nucleotide sequence with a different nucleotide sequence, resulting in the coding gene being unable to properly express a functional protein, or only able to express a protein with reduced activity. Therefore, in this paper, "disruption" of a coding gene includes gene knockout and gene mutation.

[0077] "Homology" or "identity" refers to the sequence similarity between two polypeptides or two nucleic acid sequences. The percentage of identity can be determined by comparing positions in each sequence, which can be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are identical at that position. The degree of identity between sequences depends on the number of shared matching positions. Tools for comparing sequence similarity are well known to those skilled in the art; for example, the alignment and percentage of sequence identity of the nucleic acid or amino acid sequences provided herein can be obtained by importing the nucleic acid or amino acid sequence into ClustalW (available from https: / / genome.jp / toolsbin / clustalw / ) and using ClustalW.

[0078] In this document, the terms "homologous protein" and "homological protein" are used interchangeably. Both refer to proteins that share a high degree of sequence identity in their amino acid or coding sequences with a reference protein, and that share a high degree of functional similarity, such as performing the same function or catalyzing the same reaction process. "Homologous protein" includes proteins that share at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with a reference protein (such as the 3β-hydroxysteroid dehydrogenase described herein). Alternatively, “homological protein” includes a protein encoded by a polynucleotide having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with a reference protein (e.g., the coding sequence of 3β-hydroxysteroid dehydrogenase described herein).

[0079] "Non-homologous protein" or "irrelevant protein" has less than 40%, less than 30%, or less than 25% identity with the amino acid sequence or coding sequence of the reference protein described in this invention.

[0080] As used herein, the term "promoter" refers to an expression control sequence that controls the initiation and efficiency of gene or transgene transcription. Promoters can be, for example, constitutive, inducible, repressive, or tissue-specific. Promoters may contain genetic elements that regulate proteins and molecules such as RNA polymerases and transcription factors to which they can bind. In this context, a "weak promoter" is a promoter whose ability to initiate gene transcription is reduced in a given host cell compared to a wild-type promoter in the coding gene.

[0081] When applied to specific microorganisms, the term "engineered" refers to the artificial intervention that gives the microorganism certain different characteristics compared to the original strain. For example, the engineered mycobacteria described in this invention have certain genes disrupted to enable them to synthesize desired sterol derivatives. Therefore, those skilled in the art will understand that "engineered microorganisms" are not limited to strains modified through genetic engineering; strains containing the gene disruption described in this invention, obtained through UV mutagenesis or chemical mutagenesis, should also be included within the scope of "engineered microorganisms" as described in this invention.

[0082] Proteins can be classified based on their sequence and structural characteristics. Existing protein categories typically include proteins with known functions. By analyzing the category to which a protein belongs, the function of a new protein can be predicted.

[0083] Proteins can be classified according to protein families. A protein family is a collection of proteins that share a common evolutionary origin, similar amino acid sequences, and similar structures and functions. Members of the same protein family typically have similar structures, functions, and biological significance. Protein families are usually arranged in a hierarchical structure; proteins within a family that share a common evolutionary ancestor are further divided into smaller, more closely related families, such as superfamilies, families, and subfamilies.

[0084] Proteins can also be classified according to their domains. A domain is a structural or functional unit of a protein. A protein can contain multiple domains. Different domains have unique spatial conformations and perform different biological functions. Similar domains can be distributed in different proteins. Therefore, proteins can also be classified according to their domains (domain families). Domain classification is done on a domain-by-domain basis, not on the entire protein. However, domain families and protein families often overlap.

[0085] Proteins can also be classified based on sequence characteristics. These sequence characteristics include active sites, binding sites, post-translational modification sites, and repetitive sequences.

[0086] Sterols are a class of cyclopentane polyhydrophenanthrene derivatives composed of three hexane rings and one cyclopentane ring, characterized by the presence of a hydroxyl group at the carbon 3 position. The basic skeleton of sterols can have various substituents, representative ones including a methyl group at C-10, a methyl group at C-13, and a hydrocarbon group at C-17. The hydrocarbon group at C-17 can be, for example, straight-chain, branched, or cyclic, and can be saturated or unsaturated. The cyclopentane polyhydrophenanthrene ring skeleton can contain one or more double bonds.

[0087] Sterols include animal sterols, phytosterols, and fungal sterols. "Animal sterols," "phytosterols," and "fungal sterols" refer to sterols found in animals, plants, and fungi, respectively, or sterols derived from animals, plants, and fungi. The term "plant" as used herein also includes algae.

[0088] Examples of animal sterols include, but are not limited to, cholesterol, 7-dehydrocholesterol, cholesterol alkyl, and lanosterol.

[0089] Examples of phytosterols include, but are not limited to, stigmasterol, β-sitosterol, brassicasterol, and campesterol. In this document, phytosterols also include phytosterols, including but not limited to β-sitostanol and campestanol.

[0090] Examples of fungal sterols include, but are not limited to, fucosterol and ergosterol.

[0091] Sterols can exist in, for example, free form, fatty acid ester form, glycoside form, etc. That is, unless otherwise stated, "sterol" as used herein may refer to free sterol, sterol fatty acid ester, sterol glycoside, or a mixture thereof.

[0092] The term "sterol derivative" refers to a compound formed from a parent compound molecule (i.e., a sterol) through one or more chemical reactions. "Sterol derivative in the form of 5-en-3-ol" refers to a sterol derivative having a hydroxyl group at the C-3 position and a carbon-carbon double bond between the C-5 and C-6 positions.

[0093] In this invention, unless otherwise specified, the carbon (C) positions in the sterol ring are numbered as shown in Figure 1A. For example, the terms "position 3", "position 4", and "position 5" used in this invention refer to the positions of carbon (C) 3, 4, or 5 in the sterol ring as shown in Figure 1A.

[0094] Chemical definition

[0095] The definitions of specific functional groups and chemical terms are described in more detail below.

[0096] When listing a range of values, it is assumed that each value and a subrange within that range will be included. For example, "C 1-6 Alkyl groups include C1, C2, C3, C4, C5, C6, and C6. 1-6 C 1-5 C 1-4 C 1-3 C 1-2 C 2-6 C 2-5 C 2-4 C 2-3 C 3-6 C 3-5 C 3-4 C 4-6 C 4-5 and C 5-6 alkyl.

[0097] “C 1-16 "Alkyl" refers to a straight-chain or branched saturated hydrocarbon group having 1 to 16 carbon atoms. 4-12 "Alkyl" refers to a straight-chain or branched saturated hydrocarbon group having 4 to 12 carbon atoms. 1-6 "Alkyl" refers to a straight-chain or branched saturated hydrocarbon group having 1 to 6 carbon atoms. In some embodiments, C 1-4 Alkyl groups are preferred. C 1-6 Examples of alkyl groups include: methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5), and n-hexyl (C6). The term "C" is used in conjunction with the preceding text. 1-6 "Alkyl" also includes heteroalkyl, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkyl group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. Common alkyl abbreviations include: Me(-CH3), Et(-CH2CH3), iPr(-CH(CH3)2), nPr(-CH2CH2CH3), n-Bu(-CH2CH2CH2CH3) or i-Bu(-CH2CH(CH3)2).

[0098] “C 2-16 "Alkenyl" refers to a straight-chain or branched hydrocarbon group having 2 to 16 carbon atoms and at least one carbon-carbon double bond. 2- "6-olefin" refers to a straight-chain or branched hydrocarbon group having 2 to 6 carbon atoms and at least one carbon-carbon double bond. In some embodiments, C 2-10 Alkenyl groups are preferred. C2-6 Examples of alkenyl groups include: vinyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), pentenyl (C5), pentadienyl (C5), hexenyl (C6), and so on. The term "C" is used in conjunction with these groups. 2-6 "Alkenyl" also includes heteroalkenyl groups, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkenyl group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0099] “C 2-16 "Alkyne" refers to a straight-chain or branched hydrocarbon group having 2 to 16 carbon atoms, at least one carbon-carbon triple bond, and optionally one or more carbon-carbon double bonds. 2-6 "Alkyne" refers to a straight-chain or branched hydrocarbon group having 2 to 6 carbon atoms, at least one carbon-carbon triple bond, and optionally one or more carbon-carbon double bonds. In some embodiments, C 2-10 The alkynyl group is preferred. C 2-6 Examples of alkynyl groups include, but are not limited to: ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), pentyynyl (C5), hexynyl (C6), etc. The term "C" is used in conjunction with other alkynyl groups. 2-6 "Alkyne" also includes heteroyne, wherein one or more (e.g., 1, 2, 3 or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkynyl group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents or 1 substituent.

[0100] “C 1-16 "Alkoxy" refers to the group -OR, where R is a substituted or unsubstituted carbon group. 1-16 Alkyl group. In some embodiments, C 1-6 Alkoxy groups are particularly preferred. Specific alkoxy groups include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexyloxy, and 1,2-dimethylbutoxy. The alkoxy group may be optionally substituted with one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0101] "Halogen" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0102] Therefore, "C" 1-16 "Halogenated alkyl" refers to the above "C 1-16"alkyl" is substituted with one or more halogen groups. In some embodiments, C 1-12 Haloalkyl groups are particularly preferred, and C4 groups are more preferred. 1-8 Halogenated alkyl groups. In some embodiments, C 1-6 Halogenated alkoxy groups are particularly preferred, and C4 is more preferred. 1-4 Haloalkoxy groups. Exemplary haloalkyl groups include, but are not limited to: -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, 2,2,2-trifluoro-1,1-dimethyl-ethyl, etc. Exemplary haloalkoxy groups include, but are not limited to: -OCH2F, -OCHF2, -OCF3, etc. The haloalkyl and haloalkoxy groups can be substituted at any available connection point, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0103] The carbonyl group, whether used alone or in combination with other terms (such as aminocarbonyl), is represented as -C(O)-.

[0104] "Oxyto" means =O.

[0105] "Thio" means =S.

[0106] The alkyl, alkenyl, ynyl, carbocyclic, and heteroalkyl groups (heteroalkyl, heteroalkenyl, heteroynyl) defined in this article are optional substituted groups.

[0107] In this document, compounds are named using standard nomenclature. For compounds with asymmetric centers, it should be understood (unless otherwise stated) that all optical isomers and mixtures thereof are included. Furthermore, unless otherwise specified, all isomers included in this invention may have carbon-carbon double bonds in the forms of Z and E. Regarding compounds existing in different tautomeric forms, a single compound is not limited to any particular tautomer, but is intended to encompass all tautomeric forms.

[0108] The compounds of this invention may include one or more asymmetric centers and therefore may exist in a variety of stereoisomeric forms, such as enantiomers and / or diastereomers. For example, the compounds of this invention may be individual enantiomers, diastereomers, or geometric isomers (e.g., cis and trans isomers), or may be in the form of mixtures of stereoisomers, including racemic mixtures and mixtures rich in one or more stereoisomers. This invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)- isomers, (L)- isomers, and their racemic mixtures and other mixtures, such as mixtures enriched with enantiomers or diastereomers, all of which are within the scope of this invention. In this document, “*” and “#” denote chiral centers, which may be in (S) absolute configuration, or (R) absolute configuration, or a mixture of both, i.e., racemic forms. The racemic form, also known as the racemic mixture, is used interchangeably with "racemic mixture" or "compound in racemic form," referring to an optically inactive mixture consisting of equal amounts of two opposing enantiomers. Isomers can be separated from the mixture by methods known to those skilled in the art, including chiral high-performance liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric synthesis.

[0109] The compounds of this invention can exist as tautomers. Tautomers are functional group isomers that arise from the rapid movement of an atom between two positions in a molecule. Tautomers are a special type of functional group isomer. A pair of tautomers can interconvert, but usually the more stable isomer is the dominant form. The most important examples are enol and keto tautomers.

[0110] engineered microorganisms

[0111] This invention demonstrates for the first time that MFT plays an important role in the sterol epoxidation reaction during the bioconversion of phytosterols to 4-androstenedione. By inhibiting the expression and / or activity of MFT-related proteins, sterol epoxidation can be effectively suppressed, promoting the synthesis of sterol derivatives in the form of 5-en-3-ol.

[0112] This invention uses *Mycobacterium aureum* as an example to successfully obtain engineered microorganisms capable of synthesizing DHEA from phytosterols by disrupting the gene encoding the MFT-related protein. Those skilled in the art will understand that the strains and disrupted genes used in the embodiments of this invention are merely exemplary, and the related principles can be applied to other microbial strains and other genes.

[0113] To achieve the biosynthesis of sterol derivatives in the form of 5-en-3-ol using sterols, the present invention provides an engineered microorganism whose expression and / or activity of cholesterol oxidase and / or 3β-hydroxysteroid dehydrogenase / isomerase are inhibited, and whose expression and / or activity of one or more redox cofactor mycofactocin (MFT)-related proteins are inhibited.

[0114] Many microorganisms can degrade sterols, such as Gram-negative bacteria Commonas testosteroni and Sterolibacterium denitrificans; and Gram-positive bacteria, such as Gordonia, Mycobacterium, Nocardia and Rhodococcus (Kreit, Microbial catabolism of sterols: focus on the enzymes that transform the sterol 3β-hydroxy-5-en into 3-keto-4-en. FEMS Microbiol Lett. 364(3)(2017)). For example, some microorganisms can convert cholesterol or phytosterols into AD or AD derivatives, including *Mycolicibacterium parafortuitum*, *Mycolicibacterium fortuitum*, *Mycolicibacterium vaccae*, *Mycobacterium flavum*, *Mycolicibacterium smegmatis*, *Mycolicibacterium neoaurum*, *Arthrobacter simplex* (synonym *Pimelobacter simplex*), *Rhodococcus equi*, *Brevibacterium lipolyticum*, *Nocardia ahena*, and *Pseudomonas sp. NCIB 10590*, etc. (Malaviya, A. and Gomes, J. Androstenedione production by biotransformation of phytosterols. Biores. Technol. 99, 6725-6737 (2008); Nunes, VO et al. Biotransformation of phytosterols into androstenedione—A technological prospecting study. Molecules 27, 3164 (2022)).It should be noted that in 2018, four new genera were separated from the genus *Mycobacterium*: *Mycolicibacillus*, *Mycolicibacter*, *Mycobacteroides*, and *Mycolicibacterium*. These four new genera do not yet have universally accepted Chinese translations, therefore, they will still be referred to as *Mycobacterium* in this article. Unless otherwise specified, the genus *Mycobacterium* referred to in this article includes *Mycobacterium*, *Mycolicibacillus*, *Mycolicibacter*, *Mycobacteroides*, and *Mycolicibacterium*.

[0115] Cholesterol oxidases are a class of enzymes that use flavin adenine dinucleotide (FAD) as a cofactor and molecular oxygen as a co-substrate to catalyze the conversion of the CH-OH group at the 3-position of the sterol ring to C=O and the isomerization of the double bond at the 5-position to the 4-position, while simultaneously generating hydrogen peroxide. Cholesterol oxidases are divided into two classes: one belongs to the glucose-methanol-choline (GMC) oxidoreductase family, in which the FAD cofactor is non-covalently bound to the enzyme; the other belongs to the vanillyl alcohol oxidase (VAO) family, in which the FAD cofactor is covalently bound to the enzyme. The fungi that are found in *Actinomyces lavendulae*, *Arthrobacter rhodochrous*, *Bacillus spp.*, *Arthrobacter simplex*, *Corynebacterium cholesterollicum*, *Brevibacterium sterolicum*, *Nocardia rhodochrous*, *Mycobacterium spp.*, *Pseudomonas spp.*, *Nocardia erythropolis*, *Rhodococcus equi*, *Streptomyces violascens*, *Streptomyces spp.*, *Schizophyllum commune*, *Streptomyces griseocarneus*, and *Chryseobacterium* are listed. Cholesterol oxidase has been found in microorganisms such as *Gleum* and *Gamma Proteobacterium Y-134* (Cui et al., Heterologous expression and function of cholesterol oxidase: A review. Protein Pept Lett 30, 531-540 (2023)).Taking Mycobacterium as an example, reported cholesterol oxidases include, but are not limited to, proteins encoded by the ChoD gene of Mycobacterium tuberculosis (Uniprot ID P9WMV9, SEQ ID NO:23), the ChoD homolog of Mycobacterium smegma (Uniprot ID A0QSU5, SEQ ID NO:24), the ChoD homolog of Mycobacterium aureum (GenBank: GU222349.1, SEQ ID NO:25), the protein encoded by ChoM1 of Mycobacterium aureum (GenBank: JQ303323.2, SEQ ID NO:26), and the protein encoded by ChoM2 of Mycobacterium aureum (GenBank: JQ303324.1, SEQ ID NO:27). This invention further discovered cholesterol oxidases in Mycobacterium aureum NRRL B-3805 (proteins encoded by MyAD_00355, SEQ ID NO:22 and MyAD_07380, SEQ ID NO:20) that are homologous to the above proteins.

[0116] 3β-hydroxysteroid dehydrogenase / isomerase is a class of enzymes that use NAD+. + or NADP + As a cofactor, it catalyzes the conversion of the CH-OH at position 3 of the sterol ring to C=O and the isomerization of the double bond at position 5 to position 4, simultaneously generating NADH or NADPH. 3β-hydroxysteroid dehydrogenases / isomerases are also found in various microorganisms. For example, reported 3β-hydroxysteroid dehydrogenases / isomerases include, but are not limited to, the protein encoded by the Mycobacterium tuberculosis Rv1106c gene (UniProt ID P9WQP7, SEQ ID NO:28), and the products of two genes MSMEG_5228 and MSMEG_5233 in Mycobacterium smegma (UniProt ID A0R2T6 and A0R2U1, SEQ ID NO:29 and SEQ ID NO:30, respectively). This invention further discovered a 3β-hydroxysteroid dehydrogenase / isomerase (the protein encoded by MyAD_21005, SEQ ID NO:18) in Mycobacterium neo-Aureum NRRL B-3805, which is homologous to the above protein.

[0117] Interpro is a non-redundant protein signature database that integrates 13 widely used protein classification databases (https: / / www.ebi.ac.uk / interpro / ). It classifies proteins according to protein family, domain, site, repetitive sequence, and homology superfamily (Paysan-Lafosse et al., InterPro in 2022. Nucleic Acids Res. 51, D418-D427. (2023)). Relationships between families in Interpro are displayed in the database; for example, one family may be a subfamily of another larger family, or there may be overlap between two families. The protein families or domain families mentioned in this paper refer to the smallest families to which the proteins described in the Interpro database belong.

[0118] In the Interpro system, 3β-hydroxysteroid dehydrogenase / isomerase belongs to the IPR002225 domain family (3-betahydroxysteroid dehydrogenase / isomerase (IPR002225) – InterPro entry – InterPro). For example, searching the Interpro database with amino acid sequences such as SEQ ID NO:18 and 28-30 reveals that while these sequences do not all belong to the same protein family, they all share the commonality of belonging to the IPR036291 domain family (NAD(P)-binding domain superfamily (IPR036291) – InterPro entry – InterPro). Specifically, the amino acid sequences shown in SEQ ID NO:18 and 28-29 both belong to the IPR002225 domain family. The IPR002225 domain family is a smaller domain family that overlaps with the IPR036291 domain family. Cholesterol oxidase belongs to the IPR052542 (Cholesterol Oxidase Enzyme (IPR052542)-InterPro entry-InterPro) protein family. For example, searching the InterPro database with amino acid sequences such as SEQ ID NO:20 and 22-27 reveals that these sequences all belong to the IPR052542 protein family.

[0119] Therefore, in embodiments of the present invention, the cholesterol oxidase is capable of catalyzing the conversion of the CH-OH at position 3 of the sterol ring to C=O and the isomerization of the double bond at position 5 to position 4. In some embodiments, the cholesterol oxidase is derived from the IPR052542 family. In some embodiments, the cholesterol oxidase of the present invention comprises a protein encoded by the ChoD gene of Mycobacterium tuberculosis, a ChoD homolog of Mycobacterium smegmae, a ChoD homolog of Mycobacterium neo-Aureomyces, a protein encoded by ChoM1 of Mycobacterium neo-Aureomyces, a protein encoded by ChoM2 of Mycobacterium neo-Aureomyces, a protein encoded by MyAD_00355, a protein encoded by MyAD_07380, and homologs of these proteins. Those skilled in the art will understand that other cholesterol oxidases belonging to the IPR052542 family are also applicable to the present invention.

[0120] In some embodiments, the cholesterol oxidase has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 20, 22-27. In some preferred embodiments, the cholesterol oxidase has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 20 or 22. In some more preferred embodiments, the cholesterol oxidase has the amino acid sequence shown in SEQ ID NO: 20 or 22.

[0121] In some preferred embodiments, the cholesterol oxidase is encoded by a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the nucleotide sequence shown in SEQ ID NO: 19 or 21. In some more preferred embodiments, the cholesterol oxidase is encoded by the nucleotide sequence shown in SEQ ID NO: 19 or 21.

[0122] In embodiments of the present invention, the 3β-hydroxysteroid dehydrogenase / isomerase catalyzes the conversion of the CH-OH group at position 3 of the sterol ring to C=O and the isomerization of the double bond at position 5 to position 4. In some preferred embodiments, the 3β-hydroxysteroid dehydrogenase / isomerase is derived from the IPR002225 domain family. The 3β-hydroxysteroid dehydrogenase / isomerase described in this invention includes proteins encoded by the Mycobacterium tuberculosis Rv1106c gene, the Mycobacterium smegmae MSMEG_5228 gene, the Mycobacterium smegmae MSMEG_5233 gene, the Mycobacterium neoaureum MyAD_21005 gene, and homologs of these proteins. Those skilled in the art will understand that other 3β-hydroxysteroid dehydrogenases / isomerases belonging to the IPR002225 domain family are also applicable to this invention.

[0123] In some embodiments, the 3β-hydroxysteroid dehydrogenase / isomerase has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:18. In some preferred embodiments, the 3β-hydroxysteroid dehydrogenase / isomerase has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:18. In some more preferred embodiments, the 3β-hydroxysteroid dehydrogenase / isomerase has the amino acid sequence shown in SEQ ID NO:18.

[0124] In some preferred embodiments, the 3β-hydroxysteroid dehydrogenase / isomerase is encoded by a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequence shown in SEQ ID NO:17. In some more preferred embodiments, the 3β-hydroxysteroid dehydrogenase / isomerase is encoded by the nucleotide sequence shown in SEQ ID NO:17.

[0125] MFTs belong to ribosomally synthesized and posttranslationally modified peptides (RiPPs) and are redox cofactors. Genes responsible for MFT biosynthesis typically include mftABCDEF, and these genes are usually concentrated in one region of the genome, called the MFT biosynthesis gene cluster. The biosynthesis process of MFTs can be found in "Ayikpoe et al., Occurrence, function, and biosynthesis of mycofactocin. Appl. Microbiol. Biotechnol. 103, 2903-2912. (2019); Pena-Ortiz et al., Structure elucidation of the redox cofactor mycofactocin reveals oligo-glycosylation by MftF. Chem. Sci. 11, 5182-5190. (2020)". The biosynthesis of MFT begins with the precursor peptide MftA, a ribosome-synthesized peptide with a conserved C-terminal sequence -IDGXCGVY. MftB is a molecular chaperone protein that binds to MftA. MftC is a radical S-adenosylmethionine (SAM) enzyme that catalyzes the oxidative decarboxylation and cyclization of Val and Tyr at the C-terminus of the MftA precursor peptide, generating the lactam-containing intermediate MftA*. MftE is a ferrous ion-dependent peptidase that catalyzes the hydrolysis of MftA* between Gly and Val at the C-terminus, generating the MFT precursor molecule AHDP (3-amino-5-[(phydroxyphenyl)methyl]-4,4-dimethyl-2-pyrrolidinone). MftD is a flavin mononucleotide (FMN)-dependent protein that catalyzes the oxidative deamination of AHDP, generating PMFT (pre-mycofactocin). PMFT possesses redox activity and can act as a redox cofactor. MftF is a glycosyltransferase that catalyzes the conversion of PMFT to MFT. MFT can contain a variable number of glycosyl units.In the Interpro system, MftA belongs to the IPR023988 (Mycofactocin precursor peptide IPR023988-InterPro entry-InterPro(ebi.ac.uk)) protein family; MftB belongs to the IPR023850 (Peptide chaperone MftB IPR023850-InterPro entry-InterPro(ebi.ac.uk)) protein family; MftC belongs to the IPR023913 (Mycofactocin maturase MftC IPR023913-InterPro entry-InterPro(ebi.ac.uk)) protein family; MftD belongs to the IPR023989 (Pre-mycofactocin synthase IPR023989-InterPro entry-InterPro(ebi.ac.uk)) protein family; and MftE belongs to the IPR023871 (Mycofactocin precursor peptide peptidase IPR023871-InterPro) protein family. MftF belongs to the IPR023981 (Pre-mycofactocin glycosyltransferase IPR023981-InterPro entry-InterPro(ebi.ac.uk)) protein family;

[0126] In addition to the enzymes required for the MFT biosynthesis pathway mentioned above, one or more oxidoreductase genes are usually present in the MFT synthesis gene cluster. These oxidoreductase genes are typically located within the same cis-operon as one or more genes in the MFT synthesis pathway (Ayikpoe et al., Occurrence, function, and biosynthesis of mycofactocin. Appl. Microbiol. Biotechnol. 103, 2903-2912. (2019)). The functions of these oxidoreductases have not been clearly elucidated. In a recent study, the oxidoreductase (MftG, SEQ ID NO: 76) encoded by the mftG gene in the MFT synthesis gene cluster of *Mycobacterium smegmatis* was found to play a role in circulating the redox state of MFT cofactors (Graca, AP et al. MftG is crucial for alcohol metabolism of mycobacteria by linking mycofactocin oxidation to respiration. eLife13:RP97559).

[0127] Furthermore, the MFT synthesis gene cluster typically contains a gene encoding a transcription factor, mftR, which regulates MFT biosynthesis (Mendauletova, A. and Latham, JA J Biol. Chem. 298, 101474. (2022)). For example, the MSMEG_1420 gene in *Mycobacterium smegmatis* encodes MftR (SEQ ID NO: 79), belonging to the IPR023851 family (Transcriptional regulator, TetR-type IPR023851-InterPro entry-InterPro). In *Mycobacterium neoaureum*, this transcription factor is encoded by the MyAD_06460 gene (SEQ ID NO: 77).

[0128] MFT also has counterparts with further methylation modifications. Ellerhorst et al. discovered that the enzyme catalyzing MFT methylation in *Mycobacterium smegmatis* is a SAM-dependent transmethylase encoded by mftM (SEQ ID NO:78) (Ellerhorst M. et.al. S-Adenosylmethionine(SAM)-dependent methyltransferase MftM is responsible for methylation of the redox cofactor mycofactocin. ACS Chem. Biol. 17, 3207-3217 (2022)), which belongs to the NF041255 protein family (mycofactocin oligosaccharide methyltransferase MftM NF041255-InterPro entry-InterPro). Therefore, in this paper, MFT does not refer to a single molecule, but rather to a class of molecules containing different glycosyl modifications and / or methyl modifications, including their oxidized and reduced states. However, the mftM gene is not located within the MFT synthesis gene cluster.

[0129] In this invention, the MFT synthesis gene cluster refers to a region in the genome where MFT synthesis pathway genes are concentrated. In addition to MFT synthesis pathway genes, the MFT synthesis gene cluster typically includes one or more oxidoreductase genes (which are usually located within the same cisoperon as one or more MFT synthesis pathway genes) and genes encoding transcriptional regulatory factors. Therefore, the proteins encoded by the MFT synthesis gene cluster include MFT synthesis pathway proteins (e.g., MFT precursor peptide (MftA), MftA molecular chaperone protein MftB, free radical SAM protein MftC, flavin-dependent oxidoreductase MftD, peptidase MftE, glycosyltransferase MftF), transcriptional regulatory factors (e.g., MftR), and oxidoreductases encoded by the MFT synthesis gene cluster (e.g., proteins encoded by MftG and MyAD_06490).

[0130] Bioinformatics studies have found that MFT synthesis gene clusters exist in a variety of microorganisms, including those in the phyla Actinobacteria, Proteobacteria, Chloroflexi, Euryarchaeota, and Firmicutes, with the highest distribution in the phylum Actinobacteria. Within the phylum Actinobacteria, microorganisms belonging to the gene clusters *Mycobacterium*, *Streptomyces*, *Geodermatophilus*, *Nocardiodes*, *Frankia*, *Pseudonocardia*, *Gordonia*, *Nocardia*, and *Rhodococcus* all possess the MFT synthesis gene cluster (Ayikpoe, R. et.al. Occurrence, function, and biosynthesis of mycofactocin. Appl. Microbiol. Biotechnol. 103, 2903-2912. (2019)). For example, the precursor peptide MftA for MFT synthesis can be found in nearly a thousand bacterial species in the Interpro database. Some bacterial species or strains contain both MftA (IPR023988) and cholesterol oxidase; some bacterial species or strains contain both MftA and 3β-hydroxysteroid dehydrogenase / isomerase; and some bacterial species or strains contain MftA, cholesterol dehydrogenase, and 3β-hydroxysteroid dehydrogenase / isomerase.

[0131] Accordingly, the engineered microorganisms of the present invention can originate from the phyla Actinobacteria, Proteobacteria, Chlorophyta, Archaea, or Firmicutes. In some embodiments, the engineered microorganisms of the present invention originate from the phylum Actinobacteria. In some embodiments, the engineered microorganisms of the present invention originate from the genera *Mycobacterium*, *Streptomyces*, *Geoferella*, *Nocardia*, *Frankella*, *Pseudomonas*, *Goldenella*, *Nocardia*, or *Rhodococcus*. In some preferred embodiments, the engineered microorganisms of the present invention originate from *Mycobacterium pseudoocculta*, *Mycobacterium occulta*, *Mycobacterium bovis*, *Mycobacterium flavum*, *Mycobacterium tuberculosis*, *Mycobacterium smegmatis*, or *Mycobacterium aureum*. In some preferred embodiments, the engineered microorganisms originate from *Mycobacterium aureum*. In a more preferred embodiment, the engineered microorganisms originate from the *Mycobacterium aureum* strain NRRL B-3805.

[0132] In some embodiments, the engineered microorganisms possess sterol side chain or sterol ring modification activity. In some embodiments, the engineered microorganisms naturally possess sterol side chain or sterol ring modification activity. In further embodiments, the naturally occurring sterol side chain or sterol ring modification activity of the engineered microorganisms is engineered. In some embodiments, the engineered microorganisms acquire sterol side chain or sterol ring modification activity through genetic engineering methods.

[0133] In some embodiments, the engineered microorganisms have been genetically engineered or bred to enable them to convert sterols into sterol derivatives with modified side chains. In some embodiments, the engineered microorganisms have been genetically engineered or bred to enable them to convert sterols into 4-androstenedione.

[0134] Three classes of oxidoreductases exist only in bacteria with MFT synthesis gene clusters: PF00106, PF00107, and PF00465 family oxidoreductases. These three classes of oxidoreductases are speculated to be MFT-dependent oxidoreductases (Haft, Bioinformatic evidence for a widely distributed, ribosomally produced electron carrier precursor, its maturation proteins, and its nicotinoprotein redox partners. BMC Genomics 12, 21. (2011)). Furthermore, Haft's structural and sequence analysis of the PF00106 family of oxidoreductases revealed that a sequence in this family embeds an NAD cofactor within its binding site, preventing the exchange between oxidized and reduced cofactor states. Therefore, these oxidoreductases rely on additional redox cofactors to achieve the redox cycle of their NAD cofactors (Haft, DH Mycofactocin-associated mycobacterial dehydrogenases with non-exchangeable NAD cofactors. Sci. Rep. 25:7:41074 (2017)). This further provides the molecular mechanism of MFT-dependent oxidoreductases. Based on this, Krishnamoorthy and Dubey et al. found that the alcohol dehydrogenase MSMEG_6242 in Mycobacterium smegmatis is an MFT-dependent oxidoreductase. Knocking out this dehydrogenase or the MFT synthesis gene alone would disrupt the ability of Mycobacterium smegmatis to metabolize methanol and ethanol (Krishnamoorthy et al., Mycofactocin is associated with ethanol metabolism in Mycobacteria.mBio.10,e00190-19.(2019); Dubey and Jain, Mycofactocin is essential for the establishment of methylotrophy in Mycobacterium smegmatis.Biochem.Biophys.Res.Comm.516,1073-1077.(2019)).The homologue of MSMEG_6242 in Rhodococcus jostii RHA1, MadA, has also been found to be an MFT-dependent oxidoreductase. Knocking out this dehydrogenase or the MFT synthesis gene alone would disrupt the ability of Rhodococcus to metabolize ethylene glycol and ethanol (Roccor et al., The catabolism of ethylene glycol by Rhodococcus jostii RHA1 and its dependence on mycofactocin. Appl. Environ. Microbiol. 90, e0041624. (2024)).

[0135] In this invention, the oxidoreductase MyAD_06425 (SEQ ID NO:2) was discovered, playing an important role in the sterol epoxidation reaction. Based on the sequence analysis method described in "Haft, DH Mycofactocin-associated mycobacterial dehydrogenases with non-exchangeable NAD cofactors. Sci. Rep. 25:7:41074 (2017)", MyAD_06425 contains a sequence that embeds the NAD cofactor within its binding site. Furthermore, a search of the MyAD_06425 sequence in the Interpro protein database (https: / / www.ebi.ac.uk / interpro / ) reveals it to belong to the PF00106 family, thus presuming it to be an MFT-dependent oxidoreductase.

[0136] Accordingly, the MFT-related proteins described in this invention are selected from proteins encoded by the MFT synthesis gene cluster, MFT transmethylases, and MFT-dependent oxidoreductases.

[0137] In some embodiments, the protein encoded by the MFT synthesis gene cluster is selected from: proteins from the IPR023988 family, proteins from the IPR023850 family, proteins from the IPR023913 family, proteins from the IPR023989 family, proteins from the IPR023871 family, proteins from the IPR023981 family, oxidoreductases encoded by the MFT synthesis gene cluster, and proteins from the IPR023851 family. In some preferred embodiments, the protein encoded by the MFT synthesis gene cluster is selected from: MftA, MftB, MftC, MftD, MftE, MftF, MftG, MftR, and MyAD_06490.

[0138] In some embodiments, the MFT transmethylase is selected from proteins derived from the NF041255 family. In some preferred embodiments, the MFT transmethylase is selected from MftM.

[0139] In some embodiments, the MFT-dependent oxidoreductase is derived from the PF00106, PF00107, or PF00465 family. In some preferred embodiments, the MFT-dependent oxidoreductase is derived from the PF00106 family.

[0140] In some preferred embodiments, the MFT-related protein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 76, 77, 78, and 79. In some more preferred embodiments, the MFT-related protein has the amino acid sequence shown in any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 76, 77, 78, and 79.

[0141] In some embodiments, the expression and / or activity of 3β-hydroxysteroid dehydrogenase / isomerase and one or more MFT-related proteins in the engineered microorganisms of the present invention are inhibited. In some embodiments, the expression and / or activity of cholesterol oxidase and one or more MFT-related proteins in the engineered microorganisms of the present invention are inhibited. In some embodiments, the expression and / or activity of 3β-hydroxysteroid dehydrogenase / isomerase, cholesterol oxidase, and one or more MFT-related proteins in the engineered microorganisms of the present invention are inhibited.

[0142] In some specific embodiments, the expression and / or activity of one or more of the following groups of proteins in the engineered microorganisms of the present invention are inhibited:

[0143] (1) 3β-hydroxysteroid dehydrogenase / isomerase and MFT-dependent oxidoreductase;

[0144] (2) 3β-hydroxysteroid dehydrogenase / isomerase and MftA;

[0145] (3) 3β-hydroxysteroid dehydrogenase / isomerase and MftB;

[0146] (4) 3β-hydroxysteroid dehydrogenase / isomerase and MftC;

[0147] (5) 3β-hydroxysteroid dehydrogenase / isomerase and MftD;

[0148] (6) 3β-hydroxysteroid dehydrogenase / isomerase and MftE;

[0149] (7) 3β-hydroxysteroid dehydrogenase / isomerase and MftF;

[0150] (8) Oxidoreductases encoded by 3β-hydroxysteroid dehydrogenase / isomerase and MFT synthesis gene cluster;

[0151] (9) 3β-hydroxysteroid dehydrogenase / isomerase, oxidoreductase and cholesterol oxidase encoded by the MFT synthesis gene cluster;

[0152] (10) 3β-hydroxysteroid dehydrogenase / isomerase, oxidoreductase encoded by MFT synthesis gene cluster, cholesterol oxidase, MftD and MFT-dependent oxidoreductase.

[0153] In some specific embodiments, the expression and / or activity of one or more of the following groups of proteins in the engineered microorganisms of the present invention are inhibited:

[0154] (1) A protein or a variant thereof having an amino acid sequence as shown in SEQ ID NO:18 and a protein or a variant thereof having an amino acid sequence as shown in SEQ ID NO:2;

[0155] (2) Proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:18 and proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:4;

[0156] (3) Proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:18 and proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:6;

[0157] (4) Proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:18 and proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:8;

[0158] (5) Proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:18 and proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:10;

[0159] (6) Proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:18 and proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:12;

[0160] (7) Proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:18 and proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:14;

[0161] (8) Proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:18 and proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:16;

[0162] (9) Proteins or variants thereof having an amino acid sequence as shown in SEQ ID NO:18, proteins or variants thereof having an amino acid sequence as shown in SEQ ID NO:16, proteins or variants thereof having an amino acid sequence as shown in SEQ ID NO:22 and proteins or variants thereof having an amino acid sequence as shown in SEQ ID NO:20;

[0163] (10) A protein or a variant thereof having an amino acid sequence as shown in SEQ ID NO:18, a protein or a variant thereof having an amino acid sequence as shown in SEQ ID NO:16, a protein or a variant thereof having an amino acid sequence as shown in SEQ ID NO:22, a protein or a variant thereof having an amino acid sequence as shown in SEQ ID NO:20, a protein or a variant thereof having an amino acid sequence as shown in SEQ ID NO:10 and a protein or a variant thereof having an amino acid sequence as shown in SEQ ID NO:2.

[0164] In some more specific embodiments, the expression and / or activity of one or more of the following groups of proteins in the engineered microorganisms of the present invention are inhibited:

[0165] (1) Proteins having the amino acid sequence shown in SEQ ID NO:18 and proteins having the amino acid sequence shown in SEQ ID NO:2;

[0166] (2) Proteins having the amino acid sequence shown in SEQ ID NO:18 and proteins having the amino acid sequence shown in SEQ ID NO:4;

[0167] (3) Proteins having the amino acid sequence shown in SEQ ID NO:18 and proteins having the amino acid sequence shown in SEQ ID NO:6;

[0168] (4) Proteins having the amino acid sequence shown in SEQ ID NO:18 and proteins having the amino acid sequence shown in SEQ ID NO:8;

[0169] (5) Proteins having the amino acid sequence shown in SEQ ID NO:18 and proteins having the amino acid sequence shown in SEQ ID NO:10;

[0170] (6) Proteins having the amino acid sequence shown in SEQ ID NO:18 and proteins having the amino acid sequence shown in SEQ ID NO:12;

[0171] (7) Proteins having the amino acid sequence shown in SEQ ID NO:18 and proteins having the amino acid sequence shown in SEQ ID NO:14;

[0172] (8) Proteins having the amino acid sequence shown in SEQ ID NO:18 and proteins having the amino acid sequence shown in SEQ ID NO:16;

[0173] (9) Proteins having the amino acid sequence shown in SEQ ID NO:18, proteins having the amino acid sequence shown in SEQ ID NO:16, proteins having the amino acid sequence shown in SEQ ID NO:22 and proteins having the amino acid sequence shown in SEQ ID NO:20.

[0174] (10) Proteins having the amino acid sequence shown in SEQ ID NO:18, proteins having the amino acid sequence shown in SEQ ID NO:16, proteins having the amino acid sequence shown in SEQ ID NO:22, proteins having the amino acid sequence shown in SEQ ID NO:20, proteins having the amino acid sequence shown in SEQ ID NO:10 and proteins having the amino acid sequence shown in SEQ ID NO:2.

[0175] In embodiments of the present invention, the expression and / or activity of the protein is inhibited by any one or a combination of the following methods:

[0176] (1) Destroy the protein encoding gene in the engineered microorganism;

[0177] (2) Replace the promoter of the protein-encoding gene in the engineered microorganism with a weak promoter;

[0178] (3) Introduce into the engineered microorganism a molecule that inhibits the expression and / or activity of the protein.

[0179] "Disrupting" a coding gene includes knocking out or mutating the coding gene, causing the coding gene to be unable to properly express a functional protein, or to only express a protein with reduced activity. "Disrupting" a coding gene includes disrupting the coding sequence and / or disrupting other functional elements (e.g., promoters, terminators, etc.). Methods for "disrupting" a coding gene are well known to those skilled in the art, and can be achieved, for example, through genetic engineering techniques, including homologous recombination (e.g., homologous sequence exchange with an integrative plasmid), CRISPR-based genome editing, random mutagenesis, or any combination of the above methods.

[0180] Methods for replacing gene-coding promoters with weak promoters include homologous recombination (e.g., homologous sequence exchange with integrative plasmids) and CRISPR-based genome editing techniques.

[0181] Molecules that inhibit the expression and / or activity of proteins include dsRNA, shRNA, antisense nucleic acid, small interfering RNA (siRNA), microRNA (microRNA or miRNA), antibody or its antigen-binding fragment, small molecule ligand, or combinations thereof that inhibit or silence related genes.

[0182] In some preferred embodiments, the expression and / or activity of the protein of the present invention is suppressed by disrupting the coding gene of the protein in the engineered microorganism, for example, by homologous recombination. In some more preferred embodiments, the expression and / or activity of the protein of the present invention is suppressed by deleting the coding sequence of the protein from the genome, for example, by homologous recombination.

[0183] Methods for identifying successful gene disruption are well-known to those skilled in the art, such as PCR. By designing appropriate upstream and downstream primers at the location of the disrupted gene and performing a PCR reaction, the size of the resulting PCR product fragment can reflect whether the gene has been disrupted. Alternatively, sequencing the obtained PCR product can also determine whether the gene has been disrupted.

[0184] In embodiments of the present invention, engineered microorganisms are able to synthesize sterol derivatives in the form of 5-en-3-ol using sterols.

[0185] In some embodiments, the sterol is a phytosterol, comprising a variety of naturally occurring plant-derived sterols, including β-sitosterol, campesterol, stigmasterol, and brassosterol. In some embodiments, the phytosterol also includes phytosterols, such as β-sitosterol and campesterol. In some embodiments, the sterol is an animal sterol.

[0186] In some embodiments, the sterol is selected from one or more of the following: β-sitosterol, campesterol, stigmasterol, brassosterol, β-sitosterol, campesterol, and cholesterol. In some preferred embodiments, the sterol is β-sitosterol.

[0187] In some preferred embodiments, the engineered microorganisms of the present invention are capable of synthesizing sterol derivatives in the form of 5-en-3-ol from sterols, wherein the sterol is β-sitosterol and the 5-en-3-ol sterol derivative is DHEA.

[0188] The engineered microorganisms of the present invention can synthesize derivatives thereof using compounds selected from formula (I) or formula (I'), or their tautomers, stereoisomers or racemates, and mixtures thereof:

[0189] The variables are as defined in this invention.

[0190] In some embodiments, the derivatives are selected from compounds of formula (II) or (II'), or their tautomers, stereoisomers or racemates, and mixtures thereof:

[0191] The variables are as defined in this invention.

[0192] In the compounds of formula (I) or (I'), formula (II) or (II') described in this invention, the variables are defined as follows.

[0193] In one implementation scheme It represents a single bond or a double bond.

[0194] *and#

[0195] In one implementation, * represents a chiral center selected from the (S) or (R) configuration, or its racemic form.

[0196] In one implementation, # represents a chiral center selected from the (S) or (R) configuration, or its racemic form.

[0197] R1 and R2

[0198] In one implementation, R1 is H, halogen, or C. 1-16 Alkyl, C 1-16 Haloalkyl, C 1-16 Alkoxy, C 2-16 alkenyl or C 2- 16 Alkyne group; in another embodiment, R1 is H or C. 1-12 Alkyl, C 2-12 alkenyl or C2-12 Alkyne group; in another embodiment, R1 is C 4-12 Alkyl or C 4-12 Alkenyl group.

[0199] In one implementation, R1 is not replaced; in another implementation, R1 is replaced by one, two, three, four, or five R*.

[0200] In a specific implementation plan, R1 is

[0201] In one implementation, R2 is H, halogen, or C. 1-16 Alkyl, C 1-16 Haloalkyl, C 1-16 Alkoxy, C 2-16 alkenyl or C 2- 16 Alkyne group; in another embodiment, R2 is H, halogen, C 1-6 Alkyl or C 1-6 Halogenated alkyl; in another embodiment, R2 is H or C. 1-6 Alkyl group, preferably H.

[0202] In one implementation, R2 is not replaced; in another implementation, R2 is replaced by 1, 2, 3, 4, or 5 R*.

[0203] In one implementation, R1 and R2 are not both H.

[0204] R*

[0205] In one implementation, each R* is independently selected from H, halogen, NH2, OH, C. 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1- 6-alkoxy group; in another embodiment, each R* is independently selected from H, halogen, OH, C. 1-6 Alkyl or C 1-6 Halogenated alkyl group; in another embodiment, each R* is independently selected from H, OH or C. 1-4 Alkyl groups, such as CH3 or CH2CH3.

[0206] R3 and R4

[0207] In one implementation, R3 is selected from H, halogen, OR a NR b R c C(O)R a C 1-16 Alkyl, C 1-16 Haloalkyl, C2-16 alkenyl or C 2-16 Alkyne group; in another embodiment, R3 is selected from H, halogen, OR a C(O)R a C 1-16 Alkyl or C 2-16 Alkenyl; in another embodiment, R3 is selected from OH, C(O)C 1-6 Alkylene, C 4-12 Alkyl or C 4-12 Alkenyl group.

[0208] In one implementation, R3 is not replaced; in another implementation, R3 is replaced by one, two, three, four, or five R#s.

[0209] In one specific implementation scheme, R3 is selected from OH, C(O)CH3,

[0210] In one implementation, R4 is selected from H, halogens, OR a NR b R c C(O)R a C 1-16 Alkyl, C 1-16 Haloalkyl, C 2-16 alkenyl or C 2-16 Alkyne group; in another embodiment, R4 is selected from H, halogen, OR a Or C 1-6 Alkyl; in another embodiment, R4 is selected from H, OH or C. 1-6 Alkyl group, preferably H or OH.

[0211] In one implementation, R4 is not replaced; in another implementation, R4 is replaced by one, two, three, four, or five R#s.

[0212] In one implementation, R3, R4, and the carbon atoms they are attached to together form C=O or C=S.

[0213] R#

[0214] In one implementation, each R# is independently selected from H, halogen, NH2, OH, C. 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1- 6-alkoxy group; in another embodiment, each R# is independently selected from H, halogen, OH, C. 1-6 Alkyl or C 1-6 Halogenated alkyl group; in another embodiment, each R# is independently selected from H, OH or C. 1-4Alkyl groups, such as OH or CH3.

[0215] R5

[0216] In one embodiment, R5 is selected from H, halogen, NH2, OH, C. 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 Alkyl group; in another embodiment, R5 is selected from H, halogen, OH, C. 1-6 Alkyl or C 1-6 Halogenated alkyl; in another embodiment, R5 is selected from H, OH or C. 1-4 Alkyl groups, such as H or OH.

[0217] Compounds of formula (I) or (I')

[0218] In one embodiment, the compound of formula (I) is not substituted; in another embodiment, the compound of formula (I) is substituted with m R's.

[0219] In one embodiment, the compound of formula (I') is not substituted; in another embodiment, the compound of formula (I') is substituted with m R's.

[0220] Compounds of formula (II) or (II')

[0221] In one embodiment, the compound of formula (II) is not substituted; in another embodiment, the compound of formula (II) is substituted with n Rs.

[0222] In one embodiment, the compound of formula (II') is not substituted; in another embodiment, the compound of formula (II') is substituted with n R's.

[0223] m and n

[0224] In one implementation, m is selected from 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0225] In one implementation, n is selected from 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0226] R

[0227] In one embodiment, R is selected from H, halogen, NH2, OH, C. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 2-6 alkenyl or C 2-6 Alkyne group; in another embodiment, R is selected from H, halogen, NH2, OH, C 1-6 Alkyl or C 1-6 Halogenated alkyl; in another embodiment, R is selected from H or C.1-6 Alkyl groups, such as OH, CH3, or CH2CH3.

[0228] Any technical solution or any combination thereof in any of the above specific embodiments can be combined with any technical solution or any combination thereof in other specific embodiments. For example, in compounds of formula (I) or (I'), any technical solution or any combination thereof of R1 can be combined with any technical solution or any combination thereof of R2, m, * and R, etc. This invention aims to include combinations of all these technical solutions; due to space limitations, they will not be listed one by one.

[0229] In some embodiments, the engineered microorganisms of the present invention can synthesize derivatives thereof using compounds selected from formula (I) or formula (I'), or their tautomers, stereoisomers or racemates, and mixtures thereof:

[0230] in,

[0231] Represents a single bond or a double bond;

[0232] * Represents a chiral center, selected from the (S) or (R) configuration, or its racemic form;

[0233] R1 and R2 are independently selected from H, halogens, and C. 1-16 Alkyl, C 1-16 Haloalkyl, C 1-16 Alkoxy, C 2-16 alkenyl or C 2-16 The alkynyl group, wherein R1 and R2 are optionally replaced by one, two, three, four or five R* groups;

[0234] Each R* is independently selected from H, halogen, NH2, OH, C. 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 Alkoxy;

[0235] Compounds of formula (I) or (I') may optionally be substituted with m Rs;

[0236] m is selected from 0, 1, 2, 3, 4, 5, 6, 7 or 8;

[0237] R is selected from H, halogen, NH2, OH, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 2-6 alkenyl or C 2-6 alkynyl group;

[0238] Preferably,

[0239] Represents a single bond or a double bond;

[0240] * Represents a chiral center, selected from the (S) or (R) configuration, or its racemic form;

[0241] R1 is selected from H and C. 1-12 Alkyl, C 2-12 alkenyl or C 2-12 The alkynyl group is optionally substituted by one, two, three, four or five R* groups;

[0242] Each R* is independently selected from H, halogen, OH, C. 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0243] R2 is selected from H, halogens, and C. 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0244] R1 and R2 are preferably not both H;

[0245] Compounds of formula (I) or (I') may optionally be substituted with m Rs;

[0246] m is selected from 0, 1, 2, 3, 4 or 5;

[0247] R is selected from H, halogen, NH2, OH, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0248] Preferably,

[0249] Represents a single bond or a double bond, preferably a double bond;

[0250] * Represents a chiral center, selected from the (S) or (R) configuration, or its racemic form, preferably the (R) configuration;

[0251] R1 is selected from C 4-12 Alkyl or C 4-12 The alkenyl group, which is optionally substituted with one, two, or three R* groups, for example,

[0252] Each R* is independently selected from H, OH, or C. 1-4 Alkyl groups, such as CH3 or CH2CH3;

[0253] R2 is selected from H or C. 1-6 Alkyl group, preferably H;

[0254] Compounds of formula (I) are optionally substituted with m Rs;

[0255] m is selected from 0, 1, 2, or 3;

[0256] R is selected from H or C. 1-6 Alkyl groups, such as OH, CH3, or CH2CH3;

[0257] More preferably,

[0258] Compounds of formula (I) or (I') are selected from β-sitosterol. campesterol Stigmasterol rapeseed sterol β-sitosterol campesterol and cholesterol

[0259] In some embodiments, the derivatives are selected from compounds of formula (II) or (II'), or their tautomers, stereoisomers or racemates, and mixtures thereof:

[0260] in,

[0261] Represents a single or double bond, when When representing a single bond, # represents a chiral center, selected from the (S) or (R) configuration, or its racemic form;

[0262] * Represents a chiral center, selected from the (S) or (R) configuration, or its racemic form;

[0263] R3 and R4 are independently selected from H, halogens, and OR. a NR b R c C(O)R a C 1-16 Alkyl, C 1-16 Haloalkyl, C 2-16 alkenyl or C 2- 16 The alkynyl group, wherein R3 and R4 are optionally replaced by one, two, three, four or five R# groups;

[0264] Each R# is independently selected from H, halogen, NH2, OH, C. 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 Alkoxy;

[0265] R a R b and R c Independently selected from H, halogen, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0266] Alternatively, R3, R4, and the carbon atoms they are attached to can form C=O or C=S;

[0267] R5 is selected from H, halogens, NH2, OH, and C. 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 Alkoxy;

[0268] Compounds of formula (II) or (II') may optionally be substituted with n Rs;

[0269] n is selected from 0, 1, 2, 3, 4, 5, 6, 7 or 8;

[0270] R is selected from H, halogen, NH2, OH, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 2-6 alkenyl or C 2-6 alkynyl group;

[0271] Preferably,

[0272] Represents a single or double bond, when When representing a single bond, # represents a chiral center, selected from the (S) or (R) configuration, or its racemic form;

[0273] * Represents a chiral center, selected from the (S) or (R) configuration, or its racemic form;

[0274] R3 is selected from H, halogen, OR a C(O)R a C 1-16 Alkyl or C 2-16 Alkenyl groups, which are optionally substituted with 1, 2, 3, 4 or 5 R# groups;

[0275] Each R# is independently selected from H, halogen, OH, C. 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0276] R4 is selected from H, halogens, and OR. a Or C 1-6 alkyl;

[0277] Each R a Independently selected from H, halogen, or C 1-6 alkyl;

[0278] R3 and R4 are preferably not both H;

[0279] Alternatively, R3, R4, and the carbon atoms they are attached to can form C=O;

[0280] R5 is selected from H, halogens, OH, and C. 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0281] Compounds of formula (II) or (II') may optionally be substituted with n Rs;

[0282] n is selected from 0, 1, 2, 3, 4 or 5;

[0283] R is selected from H, halogen, NH2, OH, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups;

[0284] Preferably,

[0285] Represents a single or double bond, when When representing a single bond, # represents a chiral center, selected from the (S) or (R) configuration, or its racemic form;

[0286] * Represents a chiral center, selected from the (S) or (R) configuration, or its racemic form, preferably the (S) or (R) configuration;

[0287] R3 is selected from OH, C(O)C 1-6 Alkylene, C 4-12 Alkyl or C 4-12 Alkenyl groups, optionally substituted with one, two, or three R# groups, for example, OH, C(O)CH3,

[0288] Each R# is independently selected from H, OH, or C. 1-4 Alkyl groups, such as OH or CH3;

[0289] R4 is selected from H, OH, or C. 1-6 Alkyl groups, preferably H or OH;

[0290] Alternatively, R3, R4, and the carbon atoms they are attached to can form C=O;

[0291] R5 is selected from H, OH, or C. 1-4 Alkyl groups, such as H or OH;

[0292] Compounds of formula (II) may be optionally substituted with n Rs;

[0293] n is selected from 0, 1, 2, or 3;

[0294] R is selected from H or C. 1-6 Alkyl groups, such as OH, CH3, or CH2CH3;

[0295] More preferably,

[0296] Compounds of formula (II) or (II') are selected from: dehydroepiandrosterone, pregnenolone, 17-hydroxypregnenolone 7-Dehydrocholesterol, androstenediol, 25-hydroxycholesterol 22(S)-hydroxycholesterol 7β-hydroxycholesterol 20α-hydroxycholesterol and 27-hydroxycholesterol

[0297] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. The advantages and features of this invention will become clearer with this description. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the following embodiments were performed according to conventional conditions in the art, such as those described in Sambrook and Russeii et al., Molecular Cloning: A Laboratory Manual (Third Edition) (2001), CSHL Press, or according to the manufacturer's recommendations. Unless otherwise stated, the experimental materials and reagents used in the following embodiments are commercially available.

[0298] Example

[0299] Example 1. Construction and testing of a mutant strain of *Mycobacterium aureus* NRRL B-3805 based on homologs of known cholesterol oxidase and 3β-hydroxysteroid dehydrogenase / isomerase.

[0300] 1.1 Identification of candidate genes

[0301] Literature reports two enzymes capable of catalyzing sterol epoxidation and isomerization: cholesterol oxidase and 3β-hydroxysteroid dehydrogenase / isomerase. Reported mycobacterial cholesterol oxidases include the protein encoded by the ChoD gene of *Mycobacterium tuberculosis* (Uniprot ID P9WMV9, SEQ ID NO:23), its homolog in *Mycobacterium smegmatis* (Uniprot ID A0QSU5, SEQ ID NO:24), and its homolog in *Mycobacterium aureum* (GenBank: GU222349.1, SEQ ID NO:25), as well as the subsequently discovered proteins encoded by ChoM1 (GenBank: JQ303323.1, SEQ ID NO:26) and ChoM2 (GenBank: JQ303324.1, SEQ ID NO:27) of *Mycobacterium aureum*. Reported mycobacterial 3β-hydroxysteroid dehydrogenases / isomerases include proteins encoded by the Mycobacterium tuberculosis Rv1106c gene (UniProt ID P9WQP7, SEQ ID NO:28) and products of two genes (MSMEG_5228 and MSMEG_5233) in Mycobacterium smegmatis (UniProt ID A0R2T6 and A0R2U1, SEQ ID NO:29 and SEQ ID NO:30, respectively).

[0302] This invention takes *Mycobacterium aureum* as an example, and engineered the parent strain by disrupting its sterol epoxidation reaction-related genes, and verified the sterol derivative production capacity of the engineered strain.

[0303] The *Mycobacterium neoaurum* strain NRRL B-3805 (B-3805) was purchased from the ARS Culture Collection (NRRL) (https: / / nrrl.ncaur.usda.gov / ). This strain is capable of converting phytosterols into AD (Rodriguez-Garcia et al., *Complete genome sequence of 'Mycobacterium neoaurum' NRRL B-3805, an androstenedione (AD) producer for industrial biotransformation of sterols. J. Biotechnol. 224, 64-65. (2016)*). The genome sequence of strain B-3805 was referenced using the public database GenBank: CP011022.1. Based on the alignment with known cholesterol oxidase and 3β-hydroxysteroid dehydrogenase / isomerase sequences, the protein sequence (SEQ ID NO:20) encoded by the MyAD_07380 gene (SEQ ID NO:19) in the B-3805 genome has 100% identity with ChoM1 and also high sequence identity (>81%) with ChoD from Mycobacterium tuberculosis and Mycobacterium smegma. The protein sequence (SEQ ID NO:22) encoded by the MyAD_00355 gene (SEQ ID NO:21) has 99.8% identity with ChoM2. The protein sequence (SEQ ID NO:18) encoded by the MyAD_21005 gene (SEQ ID NO:17) has 72.68% sequence identity with Rv1106c and 76.69% sequence identity with the protein product of MSMEG_5228 (Figure 7). Therefore, MyAD_00355, MyAD_07380 and MyAD_21005 in the genome of strain B-3805 became the first candidate genes to be destroyed.

[0304] 1.2 Construction of gene-damaged engineered strains

[0305] Candidate genes were disrupted in B-3805 and its derivative strains using an integrative plasmid that could not reproduce within the strain (method referred to Kendall, SL and Frita, R. Construction of targeted mycobacterial mutants by homologous recombination. Methods Mol. Biol. 465, 297-310 (2009)). The backbone sequence of the integrative plasmid was derived from the pJQ200SK plasmid (sequence from Addgene website https: / / www.addgene.org / 78497 / ), synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd. This backbone sequence contained a gentamicin selection marker and a SacB anti-selection marker. Using genomic DNA as a template, 800 to 1200 bp upstream and downstream sequences of the target sequence to be deleted were amplified by PCR. The upstream and downstream homologous sequences were then inserted into the pJQ200SK plasmid using conventional molecular biology methods to generate the integrative plasmid. A map of a representative integrative plasmid (SEQ ID NO: 75) used to disrupt MyAD_21005 is shown in Figure 3. The only difference between an integrative plasmid used to disrupt other genes and a representative plasmid is the upstream and downstream homologous sequences.

[0306] For each gene deletion, the corresponding integrative plasmid was transformed into B-3805 or a derivative strain using standard electroporation, plated onto solid agar medium containing gentamicin, and incubated at 30°C for several days. Then, single colonies were inoculated into fresh pre-seed medium containing gentamicin in 96-well plates and incubated at 30°C in a shaker for three days. For reverse selection, the cultures were diluted with sterile water and plated onto solid agar medium containing sucrose, and incubated at 30°C for four days. Several colonies were inoculated into fresh pre-seed medium in 96-well plates and incubated at 30°C in a shaker for three days. The resulting cultures were used as templates for PCR to determine if they contained the expected marker-free gene deletion. After successful reverse selection, the resulting new strains were used to generate further gene-deleted strains following the same procedure.

[0307] 1.3 Incubation of strain in 96-well plates and product production

[0308] Prepare pre-seed medium (TB medium, containing 12 g / L tryptone, 24 g / L yeast extract, 4 mL / L glycerol, 0.231 g / L KH₂PO₄, 16.43 g / L K₂HPO₄·3H₂O) and seed medium (MYC / 02 liquid medium, containing 2 g / L citric acid, 0.05 g / L ferric ammonium citrate, 0.655 g / L K₂HPO₄·3H₂O, 0.5 g / L MgSO₄·7H₂O, 2.67 g / L NH₄Cl, 10 g / L glucose, pH 7.5). Inoculate three to six colonies of the new strain produced by the above method, along with a suitable control strain, into the wells of a 96-well plate containing 500 μL of fresh pre-seed medium per well. Incubate the 96-well plate in a shaker at 30°C for 3 days. Subculture the resulting culture at a 1 / 10 volume ratio into new 96-well plates containing fresh seed medium. The plate was incubated at 30°C for 1 day in a shaker. The resulting culture was transferred to a new 96-well plate containing 40 g / L (final concentration) of 2-hydroxypropyl-β-cyclodextrin (Sinopharm Reagent XW1284463553) and 2 mg of 70% β-sitosterol (Sigma 85451) per well. The plate was incubated at 30°C for 3 days in a shaker. After incubation, 5 volumes of ethyl acetate (Sinopharm Reagent 10009418) were added to the plate, and the plate was vortexed and shaken to extract the product. The plate was centrifuged, and the organic layer was transferred to a new 96-well round-bottom plate, diluted appropriately, and then analyzed by RapidFire mass spectrometry, gas chromatography, or liquid chromatography-mass spectrometry to detect the production of AD and DHEA.

[0309] The triple knockout strain with the MyAD_00355, MyAD_07380, and MyAD_21005 genes disrupted was named SS001. The upstream and downstream homologous sequences used to disrupt the genes and the identification primers are shown in Table 1. PCR identification of SS001 was performed using primers P1 / P2, P3 / P4, and P5 / P6, confirming that the MyAD_00355, MyAD_07380, and MyAD_21005 genes in the genome of strain B-3805 were successfully disrupted (Table 1).

[0310] After incubating the bacterial culture with β-sitosterol as described above, the product was detected by gas chromatography. SS001 only produced AD and did not accumulate DHEA (Figure 4).

[0311] Table 1. Gene Damage Related Information

[0312] Example 2. Further gene disruption revealed mutant strains capable of producing DHEA.

[0313] To broadly screen for genes related to sterol epoxidation, multiple genes in the B-3805 genome were disrupted based on strain SS001 using the method described in Example 1. After incubation with the strains using the plate incubation method described in Example 1, AD products were detected by RapidFire mass spectrometry as a preliminary screening method. Table 2 shows the average RapidFire peak area of ​​AD produced by three biological repeats of the partially gene-disrupted strains, normalized to SS001. It was found that the AD peak area of ​​several strains was significantly reduced. Further detection of the products of these strains by gas chromatography revealed that strain SS002 could produce DHEA up to 1.39 g / L, while the AD yield was significantly reduced compared to SS001 (Figure 4). Compared to SS001, strain SS002 further contained disruption of the MyAD_06490 gene (SEQ ID NO:15). The upstream and downstream homologous sequences used for the disrupted gene are shown in Table 4. Identification using P7 / P8 (SEQ ID NO:73 / 74) primers confirmed that the MyAD_06490 gene was successfully disrupted (Table 4).

[0314] The protein encoded by the MyAD_06490 gene (SEQ ID NO:16) has less than 25.23% sequence identity with known cholesterol oxidases or 3β-hydroxysteroid dehydrogenases / isomerases in the literature (Figure 7).

[0315] Table 2. Average normalized RapidFire peak area of ​​AD generated by strains containing different gene disruptions in the parental strain SS001.

[0316] Further gene disruption and screening based on SS002 led to the discovery of the superior strain SS003, which further increased DHEA production to 1.8 g / L and decreased AD production (Figure 4). Compared to SS002, SS003 further incorporated the disruption of two genes: MyAD_06475 (SEQ ID NO:9) and MyAD_06425 (SEQ ID NO:1). The upstream and downstream homologous sequences used for gene disruption are shown in Table 4. Identification using P9 / P10 and P11 / P12 primers confirmed the successful disruption of the MyAD_06475 and MyAD_06425 genes (Table 4).

[0317] The protein encoded by MyAD_06475 (SEQ ID NO:10) was found to be the flavin oxidoreductase MftD, which is involved in MFT biosynthesis, after sequence alignment.

[0318] The protein encoded by MyAD_06425 (SEQ ID NO:2) belongs to the PF00106 family in the Interpro database. Sequence analysis performed according to the method described in the literature (Haft, DH Mycofactocin-associated mycobacterial dehydrogenases with non-exchangeable NAD cofactors. Sci. Rep. 25:7:41074 (2017)) predicts it to be an MFT-dependent oxidoreductase.

[0319] Analysis of the amino acid sequences shows that the enzymes encoded by the two genes have less than 19% and 24.5% sequence identity with known cholesterol oxidases and 3β-hydroxysteroid dehydrogenases / isomerases in the literature, respectively (Figure 7). The MyAD_06490 gene, as previously mentioned, encodes a protein predicted to be an oxidoreductase. Because it shares the same cis-operon as MyAD_06475, and based on descriptions of the MFT synthesis gene cluster in the literature, it is speculated that it is also a member of the MFT synthesis gene cluster.

[0320] In summary, this application identified several genes that could not be identified through similarity to known enzymes. Disruption of these genes plays a crucial role in the inhibition of sterol epoxidation and DHEA production. These genes are all associated with MFT synthesis gene clusters or MFT-dependent oxidoreductases. Therefore, MFT itself or MFT-dependent oxidoreductases play a significant role in the oxidation of the sterol ring. Inhibiting MFT synthesis or the expression or function of MFT-dependent oxidoreductases can effectively suppress the oxidation of the sterol ring, thereby benefiting DHEA production.

[0321] Example 3. Identification of key gene disruption required for the conversion of phytosterols to DHEA

[0322] To further clarify the influence of the genes discovered in Examples 1 and 2, as well as other genes in the MFT synthesis gene cluster, on the sterol epoxidation reaction, this application disrupted each gene individually in the MyAD_21005 (SEQ ID NO:17), MyAD_06425 (SEQ ID NO:1), or MftA to MyAD_06490 regions (including the MftA gene, MyAD_06465, MyAD_06470, MyAD_06475, MyAD_06480, MyAD_06485, and MyAD_06490) regions of the B-3805 genome, or disrupted the entire MftA to MyAD_06490 region. The MftA gene was not labeled in the reference genome (GenBank: CP011022.1) and was deduced through sequence alignment with a known MftA protein (Uniprot ID A0QSB6) (77% sequence identity). The identification of the MFT synthetic gene cluster and the functional identification of each gene in the cluster were derived based on the gene arrangement of cisoperons in the reference genome and the family classification of the proteins encoded by each gene in the Interpro system (Table 3). The coding sequence numbers of the relevant genes and the amino acid sequence numbers of the proteins they encode are shown in Table 3. The upstream and downstream homologous sequences used to disrupt the relevant genes and the identification primers are shown in Table 4. The strain identification results are shown in Table 4.

[0323] The strains with gene disruption were screened using the incubation method described in Example 1, and the produced AD and DHEA were detected using liquid chromatography-mass spectrometry (LC-MS). The results showed that disruption of any single gene or the entire MftA to MyAD_06490 region was insufficient to enable the strains to efficiently produce DHEA. However, when the disruption of any gene in the MftA to MyAD_06490 region or the MyAD_06425 gene was combined with the disruption of MyAD_21005, *Mycobacterium virginianum* could efficiently convert phytosterols into DHEA. When MyAD_21005 was not disrupted, double knockout of other genes reduced AD production but did not lead to efficient DHEA production (Figures 5 and 6).

[0324] Table 3. Description and sequence numbers of genes to be disrupted

[0325] Table 4. Gene Damage Related Information

[0326] Although the invention has been described with reference to specific embodiments, those skilled in the art will understand that various changes can be made and equivalents can be substituted without departing from the spirit and scope of the invention. Furthermore, many modifications can be made to adapt particular circumstances, materials, compositions, methods, and method steps to the purpose, spirit, and scope of the invention. All such modifications are within the scope of the claims.

Claims

1. An engineered microorganism in which the expression and / or activity of cholesterol oxidase and / or 3β-hydroxysteroid dehydrogenase / isomerase are inhibited, and the expression and / or activity of one or more redox cofactor mycofactocin (MFT)-related proteins are inhibited.

2. The engineered microorganism of claim 1, wherein the engineered microorganism is derived from Mycobacterium, Streptomyces, Geodermatitis, Nocardia, Frankincense, Pseudomonas, Gordonia, Nocardia, or Rhodococcus. Preferably, the engineered microorganism is derived from the genus Mycobacterium; more preferably, the engineered microorganism is derived from Mycobacterium tuberculosis, Mycobacterium pseudooccurrence, Mycobacterium occurrence, Mycobacterium bovis, Mycobacterium flavum, Mycobacterium smegmatis, or Mycobacterium aureum; even more preferably, the engineered microorganism is derived from Mycobacterium aureum; and even more preferably, the engineered microorganism is derived from Mycobacterium aureum strain NRRL B-3805.

3. The engineered microorganism of claim 1 or 2, wherein the cholesterol oxidase is derived from the IPR052542 family.

4. The engineered microorganism of any one of claims 1-3, wherein the cholesterol oxidase has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 20, 22-27; Preferably, the cholesterol oxidase has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:20 or 22; More preferably, the cholesterol oxidase has the amino acid sequence shown in SEQ ID NO:20 or 22.

5. The engineered microorganism of any one of claims 1-4, wherein the 3β-hydroxysteroid dehydrogenase / isomerase is derived from the IPR002225 domain family (3-beta hydroxysteroid dehydrogenase / isomerase(IPR002225)-InterPro entry-InterPro).

6. The engineered microorganism of any one of claims 1-5, wherein the 3β-hydroxysteroid dehydrogenase / isomerase has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 18, 28-30; Preferably, the 3β-hydroxysteroid dehydrogenase / isomerase has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:18; More preferably, the 3β-hydroxysteroid dehydrogenase / isomerase has the amino acid sequence shown in SEQ ID NO:

18.

7. The engineered microorganism of any one of claims 1-6, wherein the MFT-related protein is selected from proteins encoded by the MFT synthesis gene cluster, MFT-dependent oxidoreductases, and MFT transmethylases.

8. The engineered microorganism of claim 7, wherein the protein encoded by the MFT synthesis gene cluster is selected from: proteins derived from the IPR023988 family (Mycofactocin precursor peptide (IPR023988)-InterPro entry-InterPro), proteins derived from the IPR023850 family (Peptide chaperone MftB (IPR023850)-InterPro entry-InterPro), proteins derived from the IPR023913 family (Mycofactocin maturase MftC IPR023913-InterPro entry-InterPro), proteins derived from the IPR023989 family (Pre mycofactocin synthase (IPR023989)-InterPro entry-InterPro), proteins derived from the IPR023871 family (Mycofactocin precursor peptide peptidase (IPR023871)-InterPro). The proteins are: proteins from the IPR023981 family (Pre-mycofactocin glycosyltransferase (IPR023981)-InterPro entry-InterPro), oxidoreductases encoded by the MFT synthesis gene cluster, and proteins from the IPR023851 family (Transcriptional regulator, TetR-type IPR023851-InterPro entry-InterPro); preferably, the proteins encoded by the MFT synthesis gene cluster are selected from: MftA, MftB, MftC, MftD, MftE, MftF, MftG, MftR, and MyAD_06490.

9. The engineered microorganism of claim 7 or 8, wherein the MFT-dependent oxidoreductase is derived from the PF00106, PF00107 or PF00465 family; preferably, the MFT-dependent oxidoreductase is derived from the PF00106 family.

10. The engineered microorganism according to any one of claims 7-9, wherein the MFT transmethylase is derived from the NF041255 family (mycofactocin oligosaccharide methyltransferase MftM NF041255-InterPro entry-InterPro).

11. The engineered microorganism of any one of claims 1-10, wherein the MFT-related protein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 76, 77, 78, and 79; Preferably, the MFT-related protein has an amino acid sequence shown in any one of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 76, 77, 78 and 79.

12. The engineered microorganism of any one of claims 1-11, wherein the expression and / or activity of one or more of the following groups of proteins are inhibited: (1) 3β-hydroxysteroid dehydrogenase / isomerase and MFT-dependent oxidoreductase; (2) 3β-hydroxysteroid dehydrogenase / isomerase and MftA; (3) 3β-hydroxysteroid dehydrogenase / isomerase and MftB; (4) 3β-hydroxysteroid dehydrogenase / isomerase and MftC; (5) 3β-hydroxysteroid dehydrogenase / isomerase and MftD; (6) 3β-hydroxysteroid dehydrogenase / isomerase and MftE; (7) 3β-hydroxysteroid dehydrogenase / isomerase and MftF; (8) Oxidoreductases encoded by 3β-hydroxysteroid dehydrogenase / isomerase and MFT synthesis gene cluster; (9) 3β-hydroxysteroid dehydrogenase / isomerase, oxidoreductase and cholesterol oxidase encoded by the MFT synthesis gene cluster; (10) 3β-hydroxysteroid dehydrogenase / isomerase, oxidoreductase, cholesterol oxidase, MftD and MFT-dependent oxidoreductase encoded by the MFT synthesis gene cluster.

13. The engineered microorganism of any one of claims 1-12, wherein the expression and / or activity of one or more of the following groups of proteins are inhibited: (1) A protein or a variant thereof having an amino acid sequence as shown in SEQ ID NO:18 and a protein or a variant thereof having an amino acid sequence as shown in SEQ ID NO:2; (2) Proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:18 and proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:4; (3) Proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:18 and proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:6; (4) Proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:18 and proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:8; (5) Proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:18 and proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:10; (6) Proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:18 and proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:12; (7) Proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:18 and proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:14; (8) Proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:18 and proteins or variants thereof having the amino acid sequence shown in SEQ ID NO:16; (9) Proteins or variants thereof having an amino acid sequence as shown in SEQ ID NO:18, proteins or variants thereof having an amino acid sequence as shown in SEQ ID NO:16, proteins or variants thereof having an amino acid sequence as shown in SEQ ID NO:22 and proteins or variants thereof having an amino acid sequence as shown in SEQ ID NO:20; (10) A protein or a variant thereof having an amino acid sequence as shown in SEQ ID NO:18, a protein or a variant thereof having an amino acid sequence as shown in SEQ ID NO:16, a protein or a variant thereof having an amino acid sequence as shown in SEQ ID NO:22, a protein or a variant thereof having an amino acid sequence as shown in SEQ ID NO:20, a protein or a variant thereof having an amino acid sequence as shown in SEQ ID NO:10 and a protein or a variant thereof having an amino acid sequence as shown in SEQ ID NO:

2.

14. The engineered microorganism of any one of claims 1-13, wherein the expression and / or activity of said protein is inhibited by any one or a combination of the following methods: (1) Destroy the protein encoding gene in the engineered microorganism; (2) Replace the promoter of the protein-encoding gene in the engineered microorganism with a weak promoter; (3) Introduce into the engineered microorganism a molecule that inhibits the expression and / or activity of the protein.

15. The engineered microorganism of claim 14, wherein the expression and / or activity of the protein is inhibited by disrupting the coding gene of the protein in the engineered microorganism; preferably, the coding gene of the protein is disrupted by homologous recombination.

16. The engineered microorganism of any one of claims 1-15, wherein the engineered microorganism is capable of synthesizing sterol derivatives in the form of 5-en-3-ol using sterols.

17. The engineered microorganism of claim 16, wherein the sterol is selected from β-sitosterol, campesterol, stigmasterol, campesterol, β-sitosterol, campesterol and cholesterol.

18. The engineered microorganism of claim 16 or 17, wherein the sterol derivative in the form of 5-en-3-ol is selected from: dehydroepiandrosterone (DHEA), pregnenolone, 17-hydroxypregnenolone, 7-dehydrocholesterol, androstenediol, 25-hydroxycholesterol, 22(S)-hydroxycholesterol, 7β-hydroxycholesterol, 20α-hydroxycholesterol, and 27-hydroxycholesterol.

19. A method for preparing sterol derivatives, the method comprising: (1) In the presence of sterols and under conditions suitable for producing sterol derivatives, the engineered microorganisms of any one of claims 1-18 are cultured; and / or (2) Isolate the sterol derivative from the culture of the engineered microorganism.

20. The method of claim 19, wherein the sterol is β-sitosterol and the sterol derivative is DHEA.

21. Use of the engineered microorganism of any one of claims 1-18 in the preparation of sterol derivatives.

22. The use of claim 21, wherein the sterol is β-sitosterol and the sterol derivative is DHEA.

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