Bacterium for synthesizing progesterone, construction method of bacterium, method for synthesizing progesterone by using bacterium and application of bacterium

Abstract

The present disclosure belongs to the field of biotechnology. The invention provides a bacterium for synthesizing progesterone, a construction method of the bacterium, a method for synthesizing progesterone by using the bacterium and application of the bacterium, and the bacterium has the following advantages that the biosynthesis method for synthesizing progesterone from phytosterol provided by the invention eliminates the step of chemical synthesis, reduces the use of chemical solvents and greatly reduces environmental pollution; the problem of serious pollution of a chemical solvent in a traditional process route is solved. The invention provides a bacterium for synthesizing progesterone and a construction method thereof, which can improve the yield of progesterone so as to reduce the production cost.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a bacterium for synthesizing progesterone and a construction method thereof, and a method and application of synthesizing progesterone using the bacterium. Background Art

[0002] Progesterone, also known as progesterone, is a steroid hormone secreted by the adrenal cortex, gonads, central nervous system and peripheral nervous system, and plays an important role in human reproduction. In recent years, the clinical application scope of progesterone has been continuously expanded, and the demand has been increasing.

[0003] Traditional progesterone production methods involve chemical steps using harsh chemicals and solvents, leading to high manufacturing costs and waste disposal challenges. CN105461776A discloses a method for synthesizing progesterone, which uses 3β-ethyl ether-3,5-androstadiene-17-one as a starting material, and comprises the following steps: 1) slowly adding ethyl 2-chloropropionate to 3β-ethyl ether-3,5-androstadiene-17-one (compound 02) in a solvent containing a strong base, and undergoing addition and cyclization reactions to obtain ethyl 3β-ethyl ether-3,5-androstadiene-17,20-epoxy-20-methyl-20-carboxylate (compound 03); 2) under the protection of nitrogen or an inert gas, in a solvent containing lithium chloride, subjecting ethyl 3β-ethyl ether-3,5-androstadiene-17,20-epoxy-20-methyl-20-carboxylate (compound 03) to high-temperature decarboxylation and hydrolysis reactions to obtain progesterone; this method uses irritating chemicals and solvents, resulting in high manufacturing costs and troublesome waste disposal.

[0004] With the development of biotechnology, it has become possible to use simple carbon sources (such as ethanol and glucose) to perform de novo biosynthesis of progesterone in Saccharomyces cerevisiae; however, the commercialization of this technology is hampered by low yield and low yield. Improvements in this technology require more bacterial engineering to increase the flux of this very long metabolic pathway, such as more than 30 reaction steps starting from glucose.

[0005] Recently, the direct conversion of phytosterols to progesterone using engineered mycobacteria has been successful. Due to the similarity in chemical structure between substrate and product, fewer enzymatic steps need to be engineered, and the preparation process only requires 2 steps, which suggests that it will be easier to achieve the performance indicators required for commercialization using this technology than de novo biosynthesis. However, there are currently few reports on this technology, and the yield is still low, resulting in high production costs.

[0006] The present disclosure aims to overcome the problem of low yield of progesterone synthesized by mycobacteria in the prior art, and provides a bacterium for synthesizing progesterone and a construction method thereof, and a method and application of synthesizing progesterone using the bacterium, which can increase the yield of progesterone and thus reduce production costs. Summary of the invention

[0007] The technical problem to be solved by the present disclosure is to provide a bacterium for synthesizing progesterone and a construction method thereof, and a method and application of synthesizing progesterone using the bacterium, in order to improve the progesterone yield and thus reduce the production cost.

[0008] In order to achieve the above technical objectives, the technical solution adopted by the present disclosure is:

[0009] The first aspect of the present disclosure provides a recombinant bacterium for synthesizing progesterone, wherein the recombinant bacterium is obtained by genetically modifying a starting bacterium;

[0010] Wherein, the starting bacterium is Mycobacterium neoaurum, the genetic modification is gene disruption, at least part of the genes in the recombinant bacterium are disrupted, and the disrupted genes are selected from one or a mixture of the following gene clusters:

[0011] Gene cluster 1: Gene cluster involved in fatty acid synthase II system, used to synthesize creatine;

[0012] Gene cluster 2: Gene cluster encoding the mammalian cell entry (MCE) system related to fatty acid and cholesterol transport;

[0013] Gene cluster 3: Gene cluster involved in sterol metabolism;

[0014] The recombinant bacteria is used for increasing the yield of progesterone in progesterone synthesis.

[0015] The recombinant bacteria for synthesizing progesterone disclosed in the present invention can increase the production of progesterone, thereby reducing production costs.

[0016] In one embodiment, the gene cluster one includes the kasB gene;

[0017] The gene cluster 2 includes genes related to the mce1 (mammalian cell entry) operon; preferably, the gene cluster 2 includes one or more of mam2A, lprL, mce2D, mce1C, mce1B and mce1A genes;

[0018] The gene cluster three includes one or more of the hsaA, hsaD, hsaC, hsaB and kstR genes.

[0019] In one embodiment, the kasB gene is MyAD_17955, which comprises a nucleotide sequence as shown in SEQ ID NO: 1 or having more than 90% homology thereto.

[0020] In one embodiment, the mam2A gene is MyAD_01560, which comprises a nucleotide sequence as shown in SEQ ID NO: 2 or having more than 90% homology thereto;

[0021] Preferably, the lprL gene is MyAD_01575, which comprises a nucleotide sequence as shown in SEQ ID NO: 3 or having more than 90% homology thereto;

[0022] Preferably, the mce2D gene is MyAD_01580, which comprises a nucleotide sequence as shown in SEQ ID NO: 4 or having more than 90% homology thereto;

[0023] Preferably, the mce1C gene is MyAD_01585, which comprises a nucleotide sequence as shown in SEQ ID NO: 5 or having more than 90% homology thereto;

[0024] Preferably, the mce1B gene is MyAD_01590, which comprises a nucleotide sequence as shown in SEQ ID NO: 6 or having more than 90% homology thereto;

[0025] Preferably, the mce1A gene is MyAD_01595, which comprises a nucleotide sequence as shown in SEQ ID NO: 7 or having more than 90% homology thereto.

[0026] In one embodiment, the kstR gene is MyAD_24265, which comprises a nucleotide sequence as shown in SEQ ID NO: 8 or having more than 90% homology thereto;

[0027] Preferably, the hsaB gene is MyAD_24230, which comprises a nucleotide sequence as shown in SEQ ID NO: 9 or having more than 90% homology thereto;

[0028] Preferably, the hsaC gene is MyAD_24235, which comprises a nucleotide sequence as shown in SEQ ID NO: 10 or having more than 90% homology thereto;

[0029] Preferably, the hsaD gene is MyAD_24240, which comprises a nucleotide sequence as shown in SEQ ID NO: 11 or having more than 90% homology thereto;

[0030] Preferably, the hsaA gene is MyAD_24245, which comprises a nucleotide sequence as shown in SEQ ID NO: 12 or having more than 90% homology thereto.

[0031] In one embodiment, the disrupted gene includes the kasB gene; preferably, the kasB gene is MyAD_17955, which includes a nucleotide sequence as shown in SEQ ID NO: 1 or having more than 90% homology thereto; or,

[0032] The disrupted gene includes the kstR gene; preferably, the kstR gene is MyAD_24265, which includes a nucleotide sequence as shown in SEQ ID NO: 8 or having more than 90% homology thereto; or,

[0033] The disrupted gene includes mam2A; preferably, the mam2A gene is MyAD_01560, which includes a nucleotide sequence as shown in SEQ ID NO: 2 or having more than 90% homology thereto; or,

[0034] The disrupted genes include lprL, mce2D, mce1C, mce1B and mce1A genes; preferably, the lprL, mce2D, mce1C, mce1B and mce1A genes include nucleotide sequences as shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or having more than 90% homology thereto; or,

[0035] The disrupted gene includes hsaA; preferably, the hsaA gene is MyAD_24245, which includes a nucleotide sequence as shown in SEQ ID NO: 12 or having more than 90% homology thereto; or,

[0036] The disrupted gene includes hsaADCB; the hsaADCB is a mixture of hsaA, hsaD, hsaC and hsaB genes; preferably, the hsaADCB gene includes a nucleotide sequence as shown in SEQ ID NO: 12, SEQ ID NO: 11, SEQ ID NO: 10, SEQ ID NO: 9 or a nucleotide sequence having more than 90% homology thereto.

[0037] In one embodiment, the starting bacteria is Mycobacterium novoaureum NRRL B-3805.

[0038] In one embodiment, the gene disruption is achieved by genetic engineering techniques, resulting in a decrease in the level of protein encoded by the disrupted gene;

[0039] Preferably, the gene disruption method comprises random mutation of protein sequence, Rec / ET method, CRISPR-based genome editing or a combination thereof;

[0040] Preferably, the gene disruption is by allelic exchange with an integration plasmid.

[0041] The second aspect of the present disclosure provides a method for constructing a recombinant bacterium for synthesizing progesterone as described above, the method comprising: genetically modifying a starting bacterium to obtain the recombinant bacterium as described above, so that the level of the protein encoded by the destroyed gene of the starting bacterium is reduced;

[0042] The method in which the level of the protein encoded by the destroyed gene of the starting bacteria is reduced is called gene destruction.

[0043] In one embodiment, the gene disruption is achieved by genetic engineering technology;

[0044] Preferably, the gene disruption method comprises random mutation of protein sequence, Rec / ET method, CRISPR-based genome editing or a combination thereof;

[0045] Preferably, the gene disruption is by allelic exchange with an integration plasmid.

[0046] In one embodiment, the method comprises the following steps:

[0047] Genomic DNA was used as a template to amplify the upstream and downstream sequences of the target sequence by PCR;

[0048] Conventional molecular biology methods are used to generate an integrating plasmid containing a selectable marker;

[0049] For each disrupted gene, the integration plasmid was transformed into the host bacteria using conventional methods.

[0050] Cultivating the host bacteria;

[0051] After reverse screening, the recombinant bacteria are obtained.

[0052] A third aspect of the present disclosure provides use of the aforementioned recombinant bacteria or the aforementioned method in synthesizing progesterone.

[0053] A fourth aspect of the present disclosure provides a method for synthesizing progesterone, the method comprising: inoculating the aforementioned recombinant bacteria into a seed culture medium and mixing and incubating with a phytosterol emulsion to obtain progesterone;

[0054] Alternatively, a recombinant bacterium is constructed according to the method described above, and the obtained recombinant bacterium is inoculated into a seed culture medium and mixed with a plant sterol emulsion for incubation to obtain progesterone;

[0055] Preferably, the phytosterol emulsion is a phytosterol emulsion comprising castor oil, water and glyceryl stearate;

[0056] Preferably, the incubation conditions include: an incubation temperature of 20 to 35° C. and an incubation time of 1 to 5 days;

[0057] Preferably, the phytosterol emulsion is a 1-10% phytosterol emulsion comprising castor oil, water and glyceryl stearate.

[0058] The present disclosure has the following advantages:

[0059] (1) The present disclosure provides a biosynthetic method for synthesizing progesterone from phytosterols, which eliminates the step of chemical synthesis, reduces the use of chemical solvents and greatly reduces environmental pollution, thus solving the problem of serious chemical solvent pollution in traditional process routes.

[0060] (2) The present disclosure provides gene disruption and bacteria produced thereby, which can increase progesterone production, thereby reducing production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the prior art description. Obviously, the drawings described below are some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can also be obtained based on these drawings without creative work. The drawings herein are incorporated into the specification and constitute a part of the specification, showing embodiments that conform to the specification, and are used together with the specification to explain the principles of the specification.

[0062] Figure 1 The progesterone production produced by the ΔkasB mutant of Example 3-1, the ΔkasB mutant of Example 3-2, the control bacteria of Comparative Example 1, and the control bacteria of Comparative Example 2, respectively, as measured by gas chromatography, is shown.

[0063] Figure 2 The progesterone production produced by the ΔkstR mutant of Example 4, the ΔMyAD_01560 mutant of Example 4, the ΔMyAD_01575-01595 mutant of Example 4, and the control bacteria of Comparative Example 3 are shown.

[0064] Figure 3 The progesterone production produced by the ΔhsaA mutant of Example 5, the ΔhsaADCB mutant of Example 5, and the control bacteria of Comparative Example 4, respectively, as measured by RapidFire, is shown. DETAILED DESCRIPTION

[0065] Definition and Description

[0066] In order to make it easier to understand the present disclosure, some technical and scientific terms are specifically defined below. In the present disclosure, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell and tissue culture, microbiology-related terms and laboratory operation steps used herein are terms and routine steps widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definition and explanation of the relevant terms are provided below. It should be understood that the present disclosure is not limited to specific methods, reagents, compounds, compositions or biological systems, and of course the above can be changed. It should also be understood that the terms used in the present application are only for describing specific embodiments and are not intended to be limited.

[0067] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0068] As used herein, the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, device, product or apparatus comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or apparatuses.

[0069] In the description of this document, reference is made to “some embodiments”, “some implementation schemes” or “some implementation plans”, which describe a subset of all possible embodiments, but it can be understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0070] As used herein, the term "phytosterol", also known as plant sterol, belongs to plant steroid compounds. The main components of phytosterols include sitosterol, campesterol, stigmasterol, rapeseed sterol and corresponding alkanols, all of which are based on cyclopentane perhydrophenanthrene as the main structure and contain alcohol groups. The difference between them and cholesterol in structure is that there are more side chains on C24, such as sitosterol has an ethyl group on C24, campesterol has a methyl group on C24, and the structure of stigmasterol is the same as sitosterol, except that there is a double bond on C22. Plant foods with high phytosterol content include vegetable oils, nuts and seeds, beans, etc. The phytosterol content in vegetable oils is the highest in corn germ oil, followed by sesame oil; the content of pistachios in nuts and seeds is the highest, followed by black sesame; the content of soybeans in beans is the highest, followed by green beans; the content of phytosterols in vegetables, fruits and potatoes is relatively low. The relative density of phytosterols is slightly greater than that of water. They are insoluble in water, acid and alkali, but soluble in a variety of organic solvents, such as ether, benzene, chloroform, ethyl acetate, carbon disulfide and petroleum ether.

[0071] A gene cluster refers to a gene family in which each member is tightly clustered into a large string of repeating units, located in a special region of a chromosome. A gene cluster can be composed of two adjacent related genes produced by duplication, or it can be composed of hundreds of identical genes arranged in series. A gene cluster is a product of gene amplification from the same ancestor. There are also some members of a gene family that are not tightly arranged on a chromosome and contain some unrelated sequences in between, but overall they are distributed in relatively concentrated areas on the chromosome.

[0072] Gene sequence refers to a single gene sequence or fragment. Gene is used in a broad sense, including any nucleotide sequence capable of expressing protein and any other sequence fragment.

[0073] Gene modification mainly refers to the use of biochemical methods to modify DNA sequences, introduce target gene fragments into host cells, or delete specific gene fragments from the genome, thereby changing the host cell genotype or strengthening the original genotype.

[0074] The SacB gene, or sucrose-lethal gene, is a sugar metabolism-related gene from Bacillus subtilis, and its function is to convert sucrose into fructan. The SacB gene can convert sucrose into fructans that are toxic to the host strain, so it can cause the host strain to not grow in a certain sucrose concentration and is widely used for reverse screening of certain strains. Since some strains lack genes for processing fructans, expressing the SacB gene in some strains will cause the product of the SacB gene, fructan, to aggregate in these strains, leading to the death of these strains; therefore, no other conditions are required, and only a certain concentration of sucrose needs to be added to the culture medium to achieve screening of certain strains.

[0075] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology is a method that uses RNA-guided DNA endonucleases to cut DNA at specific locations to achieve gene editing.

[0076] PCR (Polymerase chain reaction) is a nucleic acid synthesis technology that uses the principle of DNA double-strand replication to replicate specific DNA fragments in vitro.

[0077] Currently, there are two possible biosynthetic pathways for the conversion of phytosterols such as β-sitosterol into progesterone.

[0078] The figure below shows a schematic diagram of a metabolic pathway for the conversion of β-sitosterol to progesterone. The metabolic pathway is as follows: β-sitosterol is converted into β-sitostenone by cholesterol oxidase (CHO), hydroxysteroid dehydrogenase (hsdD), and ketosteroid isomerase (ksi); β-sitosterone is converted into progesterone by cytochrome P450 and redox chaperone protein (RPP). In this metabolic pathway, the side chain of β-sitosterone is cleaved by cytochrome P450 to produce progesterone; cytochrome P450 is activated by redox chaperone proteins such as ferredoxin and ferredoxin reductase.

[0079]

[0080] The figure below shows a schematic diagram of another metabolic pathway for the conversion of β-sitosterol to progesterone. This metabolic pathway is as follows: β-sitosterol is converted to pregnenolone by cytochrome P450 and redox chaperone protein (RPP); pregnenolone is converted to progesterone by cholesterol oxidase (CHO), hydroxysteroid dehydrogenase (hsdD), and ketosteroid isomerase (ksi). In this metabolic pathway, the side chain of β-sitosterol is cleaved to generate pregnenolone; pregnenolone is converted to progesterone by oxidation of the 3β-hydroxyl group and Δ5-4 isomerization.

[0081]

[0082] Detailed description of the specific implementation method

[0083] In order to achieve the above technical objectives, the technical solution adopted by the present disclosure is:

[0084] The first aspect of the present disclosure provides a recombinant bacterium for synthesizing progesterone, wherein the recombinant bacterium is obtained by genetically modifying a starting bacterium;

[0085] Wherein, the starting bacterium is Mycobacterium neoaurum, the genetic modification is gene disruption, at least part of the genes in the recombinant bacterium are disrupted, and the disrupted genes are selected from one or a mixture of the following gene clusters:

[0086] Gene cluster 1: Gene cluster involved in fatty acid synthase II system, used to synthesize creatine;

[0087] Gene cluster 2: Gene cluster encoding the mammalian cell entry (MCE) system related to fatty acid and cholesterol transport;

[0088] Gene cluster 3: Gene cluster involved in sterol metabolism;

[0089] The recombinant bacteria is used for increasing the yield of progesterone in progesterone synthesis.

[0090] The starting bacteria may be wild-type Mycobacterium neoaurea or modified Mycobacterium neoaurea. Wild-type Mycobacterium neoaurea refers to Mycobacterium neoaurea that has not been genetically modified. Modified Mycobacterium neoaurea refers to Mycobacterium neoaurea that has been genetically modified.

[0091] In one embodiment, the starting bacteria is wild-type Mycobacterium neoaureum.

[0092] In one embodiment, the starting bacteria is a modified Mycobacterium neoaurea, and is modified according to the genetic modification described in WO2024131933A1.

[0093] In one embodiment, the starting bacteria is modified Mycobacterium aureum NRRL B-3805, and is modified according to the genetic modification described in WO2024131933A1.

[0094] In one embodiment, the engineered Mycobacterium aureum expresses: (a) a non-native side-chain cleavage P450 enzyme; and (b) one or more non-native redox chaperone proteins.

[0095] In some preferred embodiments of the present disclosure, the one or more non-native redox chaperone proteins comprise a non-native ferredoxin and / or a non-native ferredoxin reductase.

[0096] In some preferred embodiments of the present disclosure, the non-native side-chain cleavage P450 enzyme is a Novosphingobium sp. PP1Y side-chain cleavage P450 enzyme or a Novosphingobium subterraneum side-chain cleavage P450 enzyme.

[0097] In certain embodiments, the one or more non-native redox chaperones include non-native ferredox proteins and / or non-native ferredox protein reductases. In certain embodiments, the non-native side chain cleavage P450 enzymes are CYP_9SPHN_1, CYP_9SPHN_2, CYP_NOVAD_1, CYP_NOVAD_2, CYP_9ACTN_2, CYP_9BACT_1, CYP_9GAMM_1, CYP_9GAMM_2, CYP_9GAMM_3, CYP_9PSEU_1, CYP_9PSEU_2, CYP_9PSEU_4 in WO2024131933A1. , CYP_9SPHN_3, CYP_9SPHN_6, CYP_9SPHN_7, CYP_XXXXX_1, CYP_XXXXX_4, CYP_XXXXX_6, CYP_XXXXX_7, CYP_XXXXX_8, CYP_XXXXX_9, CYP_XXXXX_12 have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity. In certain embodiments, the non-native ferredoxin has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with any one of FDX_SPIOL_1, camA_PSEPU_1, camA_PSEPU_2, FDX_SPIOL_2, FDX_SPIOL_3, FDX_CHLRE_1, FDX_CHLRE_2, FDX_SORC5_2, FDX_SYNY4_1, FDX_SYNE7_1, FDX_SYNE7_3, FDX_SYNE7_4, FDX_SYNE7_5, FDX_SYNE7_6, and FDX_SYNY4_2 in WO2024131933A1. In certain embodiments, the non-native ferredoxin reductase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with any one of FDXR_SPIOL_1, camB_PSEPU_1, camB_PSEPU_2, FDXR_SPIOL_2, FDXR_SPIOL_3, FDXR_SORC 5_1, FDXR_CHLR E_1, FDXR_CHLR E_2, FDXR_SYNE 7_1, FDXR_SYNE 7_2, and FDXR_SYNY 4_2 in WO2024131933A1.In certain embodiments, the non-native side chain cleavage P450 enzyme has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with a Novosphingobium sp. PP1Y side chain cleavage P450 enzyme (e.g., CYP_XXXXX_1 in WO2024131933A1) or a Novosphingobium subterraneum side chain cleavage P450 enzyme (e.g., CYP_NOVAD_1 in WO2024131933A1).

[0098] In some preferred embodiments of the present disclosure, the non-native ferredoxin is Synechococcus elongatus ferredoxin; the non-native ferredoxin reductase is Synechococcus elongatus ferredoxin reductase.

[0099] In some preferred embodiments of the present disclosure, the non-native ferredoxin is Chlamydomonas reinhardtii ferredoxin; the non-native ferredoxin reductase is Synechococcus elongatus ferredoxin reductase.

[0100] In some preferred embodiments of the present disclosure, the non-native ferredoxin is spinach (Spinacia oleracea) ferredoxin; the non-native ferredoxin reductase is spinach (Spinacia oleracea) ferredoxin reductase.

[0101] In some preferred embodiments of the present disclosure, the non-native side-chain cleavage P450 enzyme is a Novosphingobium sp. PP1Y side-chain cleavage P450 enzyme; wherein the non-native ferredoxin is a Spinacia oleracea ferredoxin; wherein the non-native ferredoxin reductase is a Spinacia oleracea ferredoxin reductase.

[0102] In certain embodiments, (a) the non-native ferredoxin has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a Synechococcus elongatus ferredoxin (e.g., FDX_SYNE7_1 in WO2024131933A1); and the non-native ferredoxin reductase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a Synechococcus elongatus ferredoxin reductase (e.g., FDXR_SYNE7_1 in WO2024131933A1); 7_1) has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity; (b) the non-native ferredoxin has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with a Chlamydomonas reinhardtii ferredoxin (e.g., FDX_CHLRE_1 in WO2024131933A1); and the non-native ferredoxin reductase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with a Synechococcus elongatus ferredoxin reductase (e.g., FDXR_CHLR E_1) has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity; or (c) the non-native ferredoxin has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with a spinach (Spinacia oleracea) ferredoxin (e.g., FDX_SPIOL_1 in WO2024131933A1); and the non-native ferredoxin reductase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with a spinach (Spinacia oleracea) ferredoxin (e.g., FDX_SPIOL_1 in WO2024131933A1); oleracea) ferredoxin reductase (e.g., FDXR_SPIOL_1 in WO2024131933A1) having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity.

[0103] In certain embodiments, the non-native side-chain cleavage P450 enzyme has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with a Novosphingobium sp. PP1Y side-chain cleavage P450 enzyme (e.g., CYP_XXXXX_1 in WO2024131933A1); in certain embodiments, the non-native ferredoxin has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with a Spinacia oleracea ferredoxin (e.g., FDX_SPIOL_1 in WO2024131933A1); in certain embodiments, the non-native ferredoxin reductase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with a Spinacia oleracea ferredoxin. oleracea) ferredoxin reductase (e.g., FDXR_SPIOL_1 in WO2024131933A1) having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity.

[0104] In some preferred embodiments of the present disclosure, the modified new golden Mycobacterium comprises an expression vector, which comprises: a first gene sequence encoding the non-native side-chain cleavage P450 enzyme, and a second gene sequence encoding the non-native ferredoxin or non-native ferredoxin reductase.

[0105] In some preferred embodiments of the present disclosure, the modified new golden Mycobacterium comprises an expression vector, which comprises: a first gene sequence encoding the non-native side-chain cleavage P450 enzyme, a second gene sequence encoding a non-native ferredoxin protein, and a third gene sequence encoding a non-native ferredoxin reductase.

[0106] In some preferred embodiments of the present disclosure, the expression vector further comprises a promoter (eg, a constitutive promoter), including but not limited to promoters Ptac or Ppms.

[0107] In some preferred embodiments of the present disclosure, the expression vector further comprises a ribosome binding site upstream of the first gene sequence, the second gene sequence and / or the third gene sequence.

[0108] In some preferred embodiments of the present disclosure, any two or all three of the first gene sequence, the second gene sequence and the third gene sequence are connected to express a fusion protein.

[0109] In certain embodiments, two of the first gene sequence, the second gene sequence, and the third gene sequence are connected to express a fusion protein. In certain embodiments, the fusion protein comprises a connecting peptide sequence that is at least 80%, 85%, 90%, 95% or 100% identical to connecting peptide 1 (connector sequence: EPPPPEPPPPLPPP) or connecting peptide 2 (connector sequence: GGGGSGGGGSGGGGS) in WO2024131933A1. In certain embodiments, the fusion protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of FDX_SPIOL_1-linker1-FDXR_SPIOL_1, FDX_SPIOL_1-linker2-FDXR_SPIOL_1, FDXR_SPIOL_1-linker2-FDX_SPIOL_1, FDX_SPIOL_1-linker2-CYP_XXXXX_1, CYP_XXXXX_1-linker2-FDX_SPIOL_1, CYP_XXXXX_1-linker1-FDX_SPIOL_1, and FDX_SPIOL_1-linker1-CYP_XXXXX_1 in WO2024131933A1.

[0110] In some preferred embodiments of the present disclosure, the fusion protein comprises a connecting peptide sequence.

[0111] In some preferred embodiments of the present disclosure, the expression vector comprises from the 5' end to the 3' end:

[0112] (a) a second gene sequence encoding a ferredoxin protein from spinach (Spinacia oleracea),

[0113] (b) a first gene sequence encoding a Novosphingobium sp. PP1Y side-chain cleavage P450 enzyme, and (c) a third gene sequence encoding a Spinacia oleracea ferredoxin reductase;

[0114] The spinach (Spinacia oleracea) ferredoxin and the Novosphingobium sp. PP1Y side chain cleavage P450 enzyme are connected to form a fusion protein via a connecting peptide.

[0115] In some preferred embodiments of the present disclosure, the expression vector comprises from the 5' end to the 3' end:

[0116] (a) a first gene sequence encoding a PP1Y side-chain cleavage P450 enzyme from Novosphingobium sp., (b) a second gene sequence encoding a ferredoxin from Spinacia oleracea, and

[0117] (c) The third gene sequence encoding the spinach (Spinacia oleracea) ferredoxin reductase.

[0118] In some preferred embodiments of the present disclosure, the expression vector comprises, from the 5' end to the 3' end,

[0119] (a) a first gene sequence encoding a PP1Y side-chain cleavage P450 enzyme of the genus Novosphingobium sp.,

[0120] (b) a second gene sequence encoding a spinach (Spinacia oleracea) ferredoxin protein, and (c) a third gene sequence encoding a spinach (Spinacia oleracea) ferredoxin reductase;

[0121] The Novosphingobium sp. PP1Y side chain cleavage P450 enzyme and the Spinacia oleracea ferredoxin are connected to form a fusion protein via a connecting peptide.

[0122] In certain embodiments, the expression vector comprises from the 5' end to the 3' end: (a) a second gene sequence encoding a non-native ferredoxin, wherein the non-native ferredoxin has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with a ferredoxin from Spinacia oleracea (e.g., FDX_SPIOL_1 in WO2024131933A1), (b) a first gene sequence encoding a non-native side chain cleavage P450 enzyme, wherein the non-native side chain cleavage P450 enzyme ... Novosphingobium sp.) PP1Y side chain cleavage P450 enzyme (e.g., CYP_XXXXX_1 in WO2024131933A1) having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity, and (c) a third gene sequence encoding a non-native ferredoxin reductase, wherein the non-native ferredoxin reductase is identical to Spinacia (Spinacia oleracea) ferredoxin reductase (e.g., FDXR_SPIOL_1 in WO2024131933A1) has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity; in certain embodiments, the non-native ferredoxin and the non-native side chain cleavage P450 enzyme are fused via a connecting peptide having the sequence shown in connecting peptide 1 in WO2024131933A1 (linker sequence: EPPPPEPPPPLPPP).

[0123] In certain embodiments, the expression vector comprises from the 5' end to the 3' end: (a) a first gene sequence encoding a non-native side chain cleavage P450 enzyme, wherein the non-native side chain cleavage P450 enzyme has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with a Novosphingobium sp. PP1Y side chain cleavage P450 enzyme (e.g., CYP_XXXXX_1 in WO2024131933A1), (b) a second gene sequence encoding a non-native ferredox protein, wherein the non-native ferredox protein ... oleracea) ferredoxin (e.g., FDX_SPIOL_1 in WO2024131933A1) having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity, and (c) a third gene sequence encoding a non-native ferredoxin reductase, wherein the non-native ferredoxin reductase is identical to Spinacia oleracea oleracea) ferredoxin reductase (e.g., FDXR_SPIOL_1 in WO2024131933A1) has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity; in certain embodiments, the non-native side chain cleavage P450 enzyme and the non-native ferredoxin are fused by a connecting peptide having the sequence shown in connecting peptide 1 in WO2024131933A1 (linker sequence: EPPPPEPPPPLPPP).

[0124] In certain embodiments, the expression vector comprises from the 5' end to the 3' end: (a) a first gene sequence encoding a non-native side chain cleavage P450 enzyme, wherein the non-native side chain cleavage P450 enzyme has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with a Novosphingobium sp. PP1Y side chain cleavage P450 enzyme (e.g., CYP_XXXXX_1 in WO2024131933A1), (b) a second gene sequence encoding a non-native ferredox protein, wherein the non-native ferredox protein ... oleracea) ferredoxin (e.g., FDX_SPIOL_1 in WO2024131933A1) having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity, and (c) a third gene sequence encoding a non-native ferredoxin reductase, wherein the non-native ferredoxin reductase is identical to Spinacia oleracea oleracea) ferredoxin reductase (e.g., FDXR_SPIOL_1 in WO2024131933A1) has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity; in certain embodiments, the non-native side chain cleavage P450 enzyme and the non-native ferredoxin are fused via a connecting peptide having the sequence shown in connecting peptide 2 in WO2024131933A1 (linker sequence: GGGGSGGGGSGGGGS).

[0125] In some preferred embodiments of the present disclosure, the modified new golden Mycobacterium comprises one or more gene knockouts, and the gene knockouts comprise: (a) smo3; (b) cyp142, smo3 and smo1; (c) cyp142, smo3, smo2 and kstD; (d) cyp142, smo3, smo2, chsH3 and kstD; (e) cyp142, smo3, smo2 and smo1; or (f) cyp142, smo3, smo2, smo1 and smo4.

[0126] In some preferred embodiments of the present disclosure, the modified Mycobacterium novoaurea further expresses cholesterol oxidase, hydroxysteroid dehydrogenase and / or ketosteroid isomerase.

[0127] As used herein, unless otherwise indicated, enzymes in the present disclosure (e.g., side chain cleavage P450 enzymes, ferredoxins, ferredoxin reductases, dehydrogenases, etc.) include wild-type enzymes and variants thereof. Variants may have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the wild-type enzyme, but the variants retain similar functions or activities, or may even have improved functions or activities. For example, the "side chain cleavage P450 enzyme" catalyzes the conversion of cholesterol to pregnenolone. The term "side chain cleavage P450 enzyme" includes wild-type side chain cleavage P450 enzymes and variants thereof, wherein the variants have substantially similar or even better functions or activities. Similarly, "ferredoxins" include wild-type ferredoxins and variants thereof, wherein the variants have substantially similar or even better functions or activities. "Feredoxin reductase" includes wild-type ferredoxin reductase and variants thereof, wherein the variant has substantially similar or better function or activity. Likewise, Novosphingobium sp. PP1Y side-chain cleavage P450 enzyme, Novosphingobium subterraneum side-chain cleavage P450 enzyme, Synechococcus elongatus ferredoxin, Synechococcus elongatus ferredoxin reductase, Chlamydomonas reinhardtii ferredoxin, Spinacia oleracea ferredoxin, Spinacia oleracea ferredoxin reductase, cholesterol oxidase, hydroxysteroid dehydrogenase, ketosteroid isomerase, thioesterase, 3α-hydroxysteroid dehydrogenase (3α-HSD), 5β-steroid reductase (5βR), 7β-hydroxylase, cytochrome P450 reductase, and the like include wild-type enzymes and their respective variants.

[0128] The term "non-native state" when referring to a polynucleotide or polypeptide is used herein to refer to a polynucleotide or polypeptide that does not naturally occur in a particular cell.

[0129] When referring to the environment of gene expression, the term "non-native state" refers to the expression of a gene in any context other than the genomic and cellular context in which it is naturally expressed. A gene expressed in a non-native state may have the same nucleotide sequence as the corresponding gene in the host cell, but may be expressed by a vector or at an integration point in the genome that is different from the native gene locus.

[0130] The term "native" is used herein to refer to a cellular component, such as a polynucleotide or polypeptide, that is naturally present in a particular cell. A native polynucleotide or polypeptide is endogenous to the cell.

[0131] The term "heterologous" is used herein to describe a polynucleotide or polypeptide introduced into a host cell. The term encompasses polynucleotides or polypeptides derived from organisms, species or strains different from the host cell, respectively. In this case, a heterologous polynucleotide or polypeptide has a sequence different from any sequence found in the same host cell. However, the term also encompasses polynucleotides or polypeptides with a sequence identical to the sequence found in the host cell, wherein the polynucleotide or polypeptide is present in a background different from the native sequence (for example, a heterologous polynucleotide can be connected to different promoters and inserted into a genomic location different from the native sequence). Therefore, "heterologous expression" encompasses the expression of a non-native sequence of a host cell, and the native expression of the native sequence of the host cell in a non-native state background.

[0132] When referring to a polynucleotide or polypeptide, the term "wild-type" refers to any polynucleotide having a nucleotide sequence, or a polypeptide having an amino acid sequence, found in a polynucleotide or polypeptide of a naturally occurring organism, regardless of the source of the molecule; that is, the term "wild-type" refers to sequence characteristics regardless of whether the molecule is purified from a natural source, purified after recombinant expression, or synthesized. The term "wild-type" is also used to refer to naturally occurring cells.

[0133] Enzymes are identified herein by the reactions they catalyze, and unless otherwise stated, enzymes refer to any polypeptide capable of catalyzing the identified reactions. Unless otherwise stated, enzymes may be derived from any organism and may have a natural or mutated amino acid sequence. As is well known, enzymes may have multiple functions and / or multiple names sometimes because of the differences in the organisms from which they are derived. The enzyme names used herein encompass orthologs, including enzymes that may have one or more additional functions or different names.

[0134] The term "variant" refers to a molecule having certain differences in base or amino acid sequence compared to a reference polynucleotide or polypeptide (naturally occurring), respectively, and these differences are artificially synthesized or naturally generated. These differences include substitutions, insertions, deletions, or any desired combination of such changes in the natural polynucleotide encoding the amino acid sequence.

[0135] The term "progesterone" refers to the 21 H 30 O2 (CAS#: 57-83-0) chemical compound.

[0136] The term "sequence identity" refers to the fact that two or more sequences are identical or have a specified percentage of identical amino acid residues or nucleotides when compared and aligned for maximum correspondence, either as measured by a sequence comparison algorithm or by visual inspection, in the context of two or more amino acid sequences or nucleotide sequences.

[0137] In order to perform sequence comparisons to determine percent identity of nucleotide or amino acid sequences, one sequence is typically used as a "reference sequence" to which a "test" sequence is compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, and if necessary, subsequence coordinates are specified, and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the specified program parameters. Sequence alignments for comparison can be performed using BLAST set to default parameters.

[0138] Generally, genes encoding enzymes can be introduced and expressed into new golden mycobacterium by conventional genetic modification techniques, so that the modified new golden mycobacterium has any active side chain cleavage P450 enzymes and redox chaperone proteins (e.g., ferredoxin and ferredoxin reductase). Suitable side chain cleavage P450 enzymes and redox chaperone proteins (e.g., ferredoxin and ferredoxin reductase) can be from any source, including plants, archaea, fungi, Gram-positive bacteria and Gram-negative bacteria.

[0139] One or more copies of any of these genes can be introduced into the selected new golden mycobacterium host cell. If more than one gene copy is introduced, these copies can have the same or different nucleotide sequences. In certain embodiments, one or two (or all) heterologous genes are expressed by a strong constitutive promoter (e.g., Ptac or Ppms). In certain embodiments, heterologous genes are expressed by an inducible promoter. Heterologous genes can be selected to be codon optimized to enhance expression in the selected new golden mycobacterium host cell.

[0140] In certain embodiments, the gene ID, source and amino acid sequence of the side chain cleavage P450 enzyme described herein are listed in the table below. For example, MBS0474250.1 is the GenBank ID of CYP_9SPHN_7 in the National Center for Biotechnology Information (NCBI) database.

[0141] Table 1: Side-chain cleavage P450 enzymes used in the present disclosure

[0142]

[0143]

[0144] In certain embodiments, the gene ID, source, and amino acid sequence of the ferredoxin proteins described herein are listed in the table below.

[0145] Table 2: Ferredoxins used in the present disclosure

[0146]

[0147]

[0148] In certain embodiments, the gene ID, source, and amino acid sequence of the ferredoxin reductases described herein are listed in the table below.

[0149] Table 3: Ferredoxin reductases used in the present disclosure

[0150]

[0151]

[0152] In certain embodiments, the modified new aurea mycobacterium described herein expresses a non-native side chain cleavage P450 enzyme (e.g., any one of the side chain cleavage P450 enzymes in Table 1). In certain embodiments, the modified new aurea mycobacterium described herein expresses one or more non-native redox chaperones, such as a non-native ferredoxin, a non-native ferredoxin reductase, or both. In certain embodiments, the non-native ferredoxin is any one of the ferredoxins in Table 2. In certain embodiments, the non-native ferredoxin reductase is any one of the ferredoxin reductases in Table 3.

[0153] The modified new golden mycobacterium described herein contains a vector suitable for expressing proteins (e.g., any combination of side chain cleavage P450 enzymes and redox chaperone proteins) in a host cell. In certain embodiments, the vector includes one or more gene sequences encoding the proteins described herein. The gene sequence can be from any source gene or source organism listed in Tables 1-3.

[0154] In certain embodiments, the vector described herein includes, from the 5' end to the 3' end: a promoter (e.g., Ptac or Ppms), a first gene sequence encoding any side chain cleavage P450 enzyme in Table 1, a second gene sequence encoding any ferredoxin in Table 2, and a third gene sequence encoding any ferredoxin reductase in Table 3. In certain embodiments, there is a ribosome binding site upstream of the first gene sequence, the second gene sequence, and the third gene sequence.

[0155] In certain embodiments, the vector comprises a Ppms promoter, a first gene sequence encoding a Novosphingobium subterraneum side-chain cleavage P450 enzyme (e.g., CYP_9SPHN_1 or CYP_9SPHN_2 in WO2024131933A1), a second gene sequence encoding a Spinacia oleracea ferredoxin (FDX_SPIOL_1 in WO2024131933A1), and a third gene sequence encoding a Spinacia oleracea ferredoxin reductase (e.g., FDXR_SPIOL_1 in WO2024131933A1). In certain embodiments, the vector comprises a Ppms promoter, a first gene sequence encoding a side-chain cleavage P450 enzyme from Novosphingobium aromaticivorans (e.g., CYP_NOVAD_1 in WO2024131933A1), a second gene sequence encoding a ferredoxin from Spinacia oleracea (FDX_SPIOL_1 in WO2024131933A1), and a third gene sequence encoding a ferredoxin reductase from Spinacia oleracea (e.g., FDXR_SPIOL_1 in WO2024131933A1). In certain embodiments, the vector comprises a Ptac promoter, a first gene sequence encoding a Novosphingobium subterraneum side-chain cleavage P450 enzyme (e.g., CYP_9SPHN_1 or CYP_9SPHN_2 in WO2024131933A1), a second gene sequence encoding a Spinacia oleracea ferredoxin (FDX_SPIOL_1 in WO2024131933A1), and a third gene sequence encoding a Spinacia oleracea ferredoxin reductase (e.g., FDXR_SPIOL_1 in WO2024131933A1).In certain embodiments, the vector comprises a Ptac promoter, a first gene sequence encoding a side chain cleavage P450 enzyme of Novosphingobium aromaticivorans (e.g., CYP_NOVAD_2 in WO2024131933A1), a second gene sequence encoding a ferredox protein of Spinacia oleracea (FDX_SPIOL_1 in WO2024131933A1), and a third gene sequence encoding a ferredox protein reductase of Spinacia oleracea (e.g., FDXR_SPIOL_1 in WO2024131933A1). In certain embodiments, the vector comprises any combination of the promoters and proteins shown in Table 7. In certain embodiments, the vector includes a Ppms promoter, a first gene sequence encoding a Novosphingobium sp. side-chain cleavage P450 enzyme (e.g., CYP_XXXXX_1 in WO2024131933A1), a second gene sequence encoding a spinach (Spinacia oleracea) ferredoxin reductase (FDX_SPIOL_1 in WO2024131933A1), and a third gene sequence encoding a spinach (Spinacia oleracea) ferredoxin reductase (e.g., FDXR_SPIOL_1 in WO2024131933A1).

[0156] In certain embodiments, any two of the first gene sequence, the second gene sequence, and the third gene sequence are connected to express a fusion protein. In certain embodiments, all three sequences in the first gene sequence, the second gene sequence, and the third gene sequence are connected to express a fusion protein. In certain embodiments, any one of the side chain cleavage P450 enzymes in Table 1 can be fused with any one of the ferredoxins in Table 2. In certain embodiments, any one of the side chain cleavage P450 enzymes in Table 1 can be fused with any one of the ferredoxins reductases in Table 3. In certain embodiments, any one of the ferredoxins in Table 2 can be fused with any one of the ferredoxins reductases in Table 3. In certain embodiments, the fusion protein described herein includes a connecting peptide sequence connecting two fused proteins. In certain embodiments, the connecting peptide sequence is at least 80%, 85%, 90%, 95% or 100% identical to the connecting peptide 1 (joint sequence EPPPPEPPPPLPPP) or the connecting peptide 2 (joint sequence GGGGSGGGGSGGGGS) in WO2024131933A1. In certain embodiments, the fusion protein described herein has an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of FDX_SPIOL_1-linker1-FDXR_SPIOL_1, FDX_SPIOL_1-linker2-FDXR_SPIOL_1, FDXR_SPIOL_1-linker2-FDX_SPIOL_1, FDX_SPIOL_1-linker2-CYP_XXXXX_1, CYP_XXXXX_1-linker2-FDX_SPIOL_1, CYP_XXXXX_1-linker1-FDX_SPIOL_1, and FDX_SPIOL_1-linker1-CYP_XXXXX_1 in WO2024131933A1. In certain embodiments, the connecting peptide described herein is a rigid connecting peptide. In certain embodiments, the connecting peptide is a flexible connecting peptide.

[0157] Successful construction of recombinant fusion proteins usually requires two elements: component proteins and connecting peptides. The selection of component proteins is based on the desired function of the fusion protein product and is relatively simple in most cases. However, selecting suitable connecting peptides to connect protein domains together can be complicated and is often overlooked in the design of fusion proteins. Direct fusion of functional domains without connecting peptides may lead to many adverse results, including misfolding of fusion proteins, lower protein yields, or impaired biological activity. Therefore, selecting or rationally designing connecting peptides that connect fusion protein domains is an important but not yet fully explored field in recombinant fusion protein technology. Details of connecting peptide design, especially the choice between flexible connecting peptides and rigid connecting peptides, can be found in, for example, Chen, X. et al. ("Fusion protein linkers: property, design and functionality", Advanced Drug Delivery Reviews ("Fusion protein linker: characteristics, design and function", Advanced Drug Delivery Reviews) 65.10 (2013): 1357-1369), the entire contents of which are incorporated herein by reference.

[0158] In certain embodiments, the vector described herein includes, from the 5' end to the 3' end, a Ppms promoter, a first sequence encoding a Novosphingobium sp. side-chain cleavage P450 enzyme (e.g., CYP_XXXXX_1 in WO2024131933A1), and a second sequence encoding a fusion protein comprising, from the N-terminus to the C-terminus, a Spinacia oleracea ferredoxin protein (e.g., FDX_SPIOL_1 in WO2024131933A1) and a Spinacia oleracea ferredoxin reductase (e.g., FDXR_SPIOL_1 in WO2024131933A1). In certain embodiments, the fusion protein has an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to FDX_SPIOL_1-linker1-FDXR_SPIOL_1 or FDX_SPIOL_1-linker2-FDXR_SPIOL_1 in WO2024131933A1.

[0159] In certain embodiments, the vector described herein includes, from the 5' end to the 3' end, a Ppms promoter, a first sequence encoding a side-chain cleavage P450 enzyme of Novosphingobium sp. (e.g., CYP_XXXXX_1 in WO2024131933A1), and a second sequence encoding a fusion protein comprising, from the N-terminus to the C-terminus, a Spinacia oleracea ferredoxin reductase (e.g., FDXR_SPIOL_1 in WO2024131933A1) and a Spinacia oleracea ferredoxin (e.g., FDX_SPIOL_1 in WO2024131933A1). In certain embodiments, the fusion protein has an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to FDXR_SPIOL_1-linker2-FDX_SPIOL_1 in WO2024131933A1.

[0160] In certain embodiments, the vector described herein comprises, from 5' to 3' end, a Ppms promoter, a first sequence encoding a fusion protein, and a second sequence encoding a spinach (Spinacia oleracea) ferredoxin reductase (e.g., FDXR_SPIOL_1 in WO2024131933A1), and the fusion protein comprises, from N-terminus to C-terminus, a spinach (Spinacia oleracea) ferredoxin (e.g., FDX_SPIOL_1 in WO2024131933A1) and a Novosphingobium sp. side-chain cleavage P450 enzyme (e.g., CYP_XXXXX_1 in WO2024131933A1). In certain embodiments, the fusion protein has an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to FDX_SPIOL_1-linker2-CYP_XXXXX_1 or FDX_SPIOL_1-linker1-CYP_XXXXX_1 in WO2024131933A1.

[0161] In certain embodiments, the vector described herein includes, from 5' to 3' end, a Ppms promoter, a first sequence encoding a fusion protein, and a second sequence encoding a spinach (Spinacia oleracea) ferredoxin reductase (e.g., FDXR_SPIOL_1 in WO2024131933A1), and the fusion protein includes, from N-terminus to C-terminus, a Novosphingobium sp. side-chain cleavage P450 enzyme (e.g., CYP_XXXXX_1 in WO2024131933A1) and a spinach (Spinacia oleracea) ferredoxin (e.g., FDX_SPIOL_1 in WO2024131933A1). In certain embodiments, the fusion protein has an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to CYP_XXXXX_1-linker2-FDX_SPIOL_1 or CYP_XXXXX_1-linker1-FDX_SPIOL_1 in WO2024131933A1.

[0162] In certain embodiments, the modified Mycobacterium neoaurum described herein contains a disruption (e.g., deletion) of one or more genes in Table 8 in the host cell genome. In certain embodiments, disruption is achieved by transforming the corresponding integration plasmid into a host Mycobacterium neoaurum cell (e.g., Mycobacterium neoaurum NRRL B-3805). In certain embodiments, the one or more genes disrupted include smo1, smo2, smo3, smo4, cyp142, kstD, chsH3, or any combination thereof. In certain embodiments, the one or more genes disrupted include cyp142, smo3, smo2, and smo1. In certain embodiments, the one or more genes disrupted include cyp142, smo3, smo2, smo1, and smo4.

[0163] Genetic modification methods:

[0164] Mycobacterium novaeum may be modified for the production of fermentative progesterone using conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry within the skill of the art. Such techniques are fully explained in the literature, see, for example, Sambrook et al., eds., Molecular Cloning: A Laboratory Manual, 4th edition, 2012; MJ Gait, ed., Oligonucleotide Synthesis, 1984); RI Freshney, ed., Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th edition, 2010; Academic Press, Inc., Current Protocols in Molecular Biology; FM Ausubel et al., eds., Current Protocols in Molecular Biology, 1987, and regularly updated; Mullis et al., eds., PCR: The Polymerase Chain Reaction, 1994; Singleton et al., eds., Dictionary of Microbiology and Molecular Biology, 1996. Biology” (Dictionary of Microbiology and Molecular Biology), 2nd edition, J. Wiley & Sons (New York, NY 1994).

[0165] Vector is a polynucleotide vector for introducing genetic material into a cell. The vector used for the methods described herein can be linear or circular. The vector can be integrated into the target genome of the host cell or replicate independently in the host cell. For many applications, an integration vector that produces a stable transformant is preferred. The vector may include, for example, an origin of replication, a multiple cloning site (MCS) and / or a selectable marker, etc. An expression vector generally includes an expression cassette that contains regulatory elements that promote the expression of a polynucleotide sequence (usually a coding sequence) in a specific host cell. Vectors include, but are not limited to, integration vectors, prokaryotic plasmids, episomes, viral vectors, cosmids, and artificial chromosomes.

[0166] Exemplary regulatory elements that can be used for expression cassettes include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals such as polyadenylation signals and poly-U sequences). Such regulatory elements are described in, for example, Goeddel, Gene Expression Technology: Methods In Enzymology, 185, Academic Press, San Diego, Calif. (1990).

[0167] In certain embodiments, the vector can be used to introduce a system capable of genome editing, such as a CRISPR system. See U.S. Patent Publication No. 2014 / 0068797 published on March 6, 2014; see also Jinek M. et al., "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity", Science, 337: 816-21, 2012. In the type II CRISPR-Cas9 system, Cas9 is a site-directed nuclease, i.e., an enzyme that is directed or can be directed to a specific target sequence and cleaves polynucleotides using two different endonuclease domains (HNH and RuvC / Rnase H-like domains). Since Cas9 is directed to its cleavage site by RNA, Cas9 can be modified to cleave DNA at any desired site. Therefore, Cas9 is also called "RNA-guided nuclease". More specifically, Cas9 associates with one or more RNA molecules, at least a portion of which hybridizes with a specific sequence in a target polynucleotide, thereby guiding Cas9 to a specific polynucleotide target. Ran, FA et al. ("In vivo genome editing using Staphylococcusaureus Cas9", Nature, 520 (7546): 186-91, 2015, Apr. 9); including all extended data) introduced the crRNA / tracrRNA sequences and secondary structures of 8 type II CRISPRCas9 systems. Cas9-like synthetic proteins are also known in the art (see U.S. patent application number 2014-0315985 published on October 23, 2014).

[0168] Vector or other polynucleotide can be introduced into new golden mycobacterium by various standard methods, for example transformation, conjugation, electroporation, nuclear microinjection, transduction, transfection (for example, lipofection-mediated or DEAE-dextrin-mediated transfection or transfection using recombinant phage virus), incubation with calcium phosphate DNA precipitate, high-speed bombardment with DNA-coated microprojectiles, and protoplast fusion. Transformants can be selected by any method known in the art. Suitable methods for selecting transformants are described in U.S. Patent Publications 2009 / 0203102, 2010 / 0048964 and 2010 / 0003716 and International Patent Publications WO2009 / 076676, WO 2010 / 003007 and WO 2009 / 132220.

[0169] The modified new Mycobacterium aureum:

[0170] The above method can be used to produce the new golden mycobacterium of transformation. Compared with the wild-type new golden mycobacterium, the new golden mycobacterium of transformation can have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more genetic changes, such as 30-100 changes. The new golden mycobacterium of transformation described in the following embodiment has one, two or three genetic changes, but those skilled in the art can design the new golden mycobacterium with additional changes according to the guidance proposed herein. In certain embodiments, compared with the wild-type new golden mycobacterium, the new golden mycobacterium of the transformation has no more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or 4 genetic changes. In various embodiments, the engineered new Mycobacterium aureum can have a number of genetic changes falling within any of the following exemplary ranges, 1-10, 1-9, 1-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-7, 3-6, 3-5, 3-4, etc.

[0171] In certain embodiments, the modified new aurea mycobacterium expresses at least two heterologous genes encoding, for example, non-native side chain cleavage P450 enzymes and / or one or more non-native redox chaperone proteins. In various embodiments, the modified new aurea mycobacterium can include and express, for example: (1) a single copy of each of the two genes, (2) two or more copies of one of the two genes, which can be the same or different, or (3) two or more copies of each of the two genes, wherein the copies of either gene can be the same or different. The same is true for other heterologous genes that can be introduced into the modified new aurea mycobacterium.

[0172] The recombinant bacteria for synthesizing progesterone disclosed in the present invention can increase the production of progesterone, thereby reducing production costs.

[0173] The gene disruption generally involves changes in gene sequence, including but not limited to gene mutation, deletion, knockout, insertion or rearrangement, etc. Preferably, the gene disruption is gene knockout.

[0174] Gene knockout is a technology that inactivates or deletes specific genes in the body through a certain pathway.

[0175] In some preferred embodiments of the present disclosure, the starting bacteria is Mycobacterium neoaurum NRRLB-3805, i.e., Mycobacterium neoaurum NRRL B-3805.

[0176] In some preferred embodiments of the present disclosure, the gene cluster one includes the kasB gene;

[0177] The gene cluster 2 includes genes related to the mce1 (mammalian cell entry) operon; preferably, the gene cluster 2 includes one or more of mam2A, lprL, mce2D, mce1C, mce1B and mce1A genes;

[0178] The gene cluster three includes one or more of the hsaA, hsaD, hsaC, hsaB and kstR genes.

[0179] In some preferred embodiments of the present disclosure, the kasB gene is MyAD_17955, which includes a nucleotide sequence as shown in SEQ ID NO: 1 or having more than 90% homology thereto.

[0180] In some preferred embodiments of the present disclosure, the mam2A gene is MyAD_01560, which includes a nucleotide sequence as shown in SEQ ID NO: 2 or having more than 90% homology thereto;

[0181] Preferably, the lprL gene is MyAD_01575, which comprises a nucleotide sequence as shown in SEQ ID NO: 3 or having more than 90% homology thereto;

[0182] Preferably, the mce2D gene is MyAD_01580, which comprises a nucleotide sequence as shown in SEQ ID NO: 4 or having more than 90% homology thereto;

[0183] Preferably, the mce1C gene is MyAD_01585, which comprises a nucleotide sequence as shown in SEQ ID NO: 5 or having more than 90% homology thereto;

[0184] Preferably, the mce1B gene is MyAD_01590, which comprises a nucleotide sequence as shown in SEQ ID NO: 6 or having more than 90% homology thereto;

[0185] Preferably, the mce1A gene is MyAD_01595, which comprises a nucleotide sequence as shown in SEQ ID NO: 7 or having more than 90% homology thereto.

[0186] In some preferred embodiments of the present disclosure, the kstR gene is MyAD_24265, which includes a nucleotide sequence as shown in SEQ ID NO: 8 or having more than 90% homology thereto;

[0187] Preferably, the hsaB gene is MyAD_24230, which comprises a nucleotide sequence as shown in SEQ ID NO: 9 or having more than 90% homology thereto;

[0188] Preferably, the hsaC gene is MyAD_24235, which comprises a nucleotide sequence as shown in SEQ ID NO: 10 or having more than 90% homology thereto;

[0189] Preferably, the hsaD gene is MyAD_24240, which comprises a nucleotide sequence as shown in SEQ ID NO: 11 or having more than 90% homology thereto;

[0190] Preferably, the hsaA gene is MyAD_24245, which comprises a nucleotide sequence as shown in SEQ ID NO: 12 or having more than 90% homology thereto.

[0191] In some preferred embodiments of the present disclosure, the disrupted gene includes the kasB gene; preferably, the kasB gene is MyAD_17955, which includes a nucleotide sequence as shown in SEQ ID NO: 1 or having more than 90% homology thereto; or,

[0192] The disrupted gene includes the kstR gene; preferably, the kstR gene is MyAD_24265, which includes a nucleotide sequence as shown in SEQ ID NO: 8 or having more than 90% homology thereto; or,

[0193] The disrupted gene includes mam2A; preferably, the mam2A gene is MyAD_01560, which includes a nucleotide sequence as shown in SEQ ID NO: 2 or having more than 90% homology thereto; or,

[0194] The disrupted genes include lprL, mce2D, mce1C, mce1B and mce1A genes; preferably, the lprL, mce2D, mce1C, mce1B and mce1A genes include nucleotide sequences as shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or having more than 90% homology thereto; or,

[0195] The disrupted gene includes hsaA; preferably, the hsaA gene is MyAD_24245, which includes a nucleotide sequence as shown in SEQ ID NO: 12 or having more than 90% homology thereto; or,

[0196] The disrupted gene includes hsaADCB; the hsaADCB is a mixture of hsaA, hsaD, hsaC and hsaB genes; preferably, the hsaADCB gene includes a nucleotide sequence as shown in SEQ ID NO: 12, SEQ ID NO: 11, SEQ ID NO: 10, SEQ ID NO: 9 or a nucleotide sequence having more than 90% homology thereto.

[0197] In some preferred embodiments of the present disclosure, the starting bacteria is Mycobacterium neoaurum NRRL B-3805.

[0198] In some preferred embodiments of the present disclosure, the gene disruption is achieved by genetic engineering technology, resulting in a decrease in the level of protein encoded by the disrupted gene;

[0199] Preferably, the gene disruption method comprises random mutation of protein sequence, Rec / ET method, CRISPR-based genome editing or a combination thereof;

[0200] Preferably, the gene disruption is by allelic exchange with an integration plasmid.

[0201] The second aspect of the present disclosure provides a method for constructing a recombinant bacterium for synthesizing progesterone as described above, the method comprising: genetically modifying a starting bacterium to obtain the recombinant bacterium as described above, so that the level of the protein encoded by the destroyed gene of the starting bacterium is reduced;

[0202] The method in which the level of the protein encoded by the destroyed gene of the starting bacteria is reduced is called gene destruction.

[0203] In some preferred embodiments of the present disclosure, the gene disruption method is achieved by genetic engineering technology; preferably, the gene disruption method includes random mutation of protein sequences, Rec / ET method, CRISPR-based genome editing or a combination thereof; preferably, the gene disruption method is allele exchange with an integrated plasmid.

[0204] In some preferred embodiments of the present disclosure, the gene disruption is achieved by genetic engineering technology.

[0205] In some preferred embodiments of the present disclosure, the gene disruption method includes random mutation of protein sequence, Rec / ET method, CRISPR-based genome editing or a combination thereof.

[0206] In some preferred embodiments of the present disclosure, the gene disruption method is allele exchange with an integration plasmid.

[0207] In the present disclosure, adding "Δ" in front of a gene indicates that the gene is knocked out. For example, the knocked-out kasB gene is ΔkasB; the mutant strain in which the kasB gene is knocked out is the ΔkasB mutant strain. The kasB gene is MyAD_17955; the ΔkasB mutant strain is the ΔMyAD_17955 mutant strain.

[0208] The kstR gene is knocked out, namely ΔkstR; the mutant strain in which the kstR gene is knocked out is the ΔkstR mutant strain. The kstR gene is MyAD_17955; the ΔkstR mutant strain is the ΔMyAD_17955 mutant strain.

[0209] The knockout of mam2A gene is Δmam2A; the mutant strain of the knockout of mam2A gene is Δmam2A mutant strain. The mam2A gene is MyAD_01560; the Δmam2A mutant strain is ΔMyAD_01560 mutant strain.

[0210] The lprL, mce2D, mce1C, mce1B and mce1A genes were knocked out, namely ΔMyAD_01575-01595; the lprL, mce2D, mce1C, mce1B and mce1A gene were knocked out, namely ΔMyAD_01575-01595 mutant strain. The lprL gene is MyAD_01575, the mce2D gene is MyAD_01580, the mce1C gene is MyAD_01585, the mce1B gene is MyAD_01590, and the mce1A gene is MyAD_01595; the lprL, mce2D, mce1C, mce1B and mce1A genes are represented by MyAD_01575-01595; the ΔMyAD_01575-01595 indicates that the lprL, mce2D, mce1C, mce1B and mce1A genes are knocked out.

[0211] The lprL gene is knocked out, namely ΔlprL; the mutant strain in which the lprL gene is knocked out is the ΔlprL mutant strain. The lprL gene is MyAD_01575; the ΔlprL mutant strain is the ΔMyAD_01575 mutant strain.

[0212] The knockout of mce2D gene is Δmce2D; the mutant strain in which the mce2D gene is knocked out is Δmce2D mutant strain. The mce2D gene is MyAD_01580; the Δmce2D mutant strain is ΔMyAD_01580 mutant strain.

[0213] The knockout of mce1C gene is Δmce1C; the mutant strain of mce1C gene knockout is Δmce1C mutant strain. The mce1C gene is MyAD_01585; the Δmce1C mutant strain is ΔMyAD_01585 mutant strain.

[0214] The knockout of mce1B gene is Δmce1B; the mutant strain of the knockout of mce1B gene is Δmce1B mutant strain. The mce1B gene is MyAD_01590; the Δmce1B mutant strain is ΔMyAD_01590 mutant strain.

[0215] The knockout of mce1A gene is Δmce1A; the mutant strain in which the mce1A gene is knocked out is Δmce1A mutant strain. The mce1A gene is MyAD_01595; the Δmce1A mutant strain is ΔMyAD_01595 mutant strain.

[0216] The hsaADCB gene is knocked out, namely ΔhsaADCB; the mutant strain in which the hsaADCB gene is knocked out is the ΔhsaADCB mutant strain. The hsaADCB is a mixture of hsaA, hsaD, hsaC and hsaB genes.

[0217] The hsaA gene is knocked out, namely ΔhsaA; the mutant strain in which the hsaA gene is knocked out is the ΔhsaA mutant strain. The hsaA gene is MyAD_24245; the ΔhsaA mutant strain is the MyAD_24245 mutant strain.

[0218] The hsaD gene is knocked out, namely ΔhsaD; the mutant strain in which the hsaD gene is knocked out is the ΔhsaD mutant strain. The hsaD gene is MyAD_24240; the ΔhsaD mutant strain is the ΔMyAD_24240 mutant strain.

[0219] The hsaC gene is knocked out, namely ΔhsaC; the mutant strain in which the hsaC gene is knocked out is the ΔhsaC mutant strain. The hsaC gene is MyAD_24235; the ΔhsaC mutant strain is the ΔMyAD_24235 mutant strain.

[0220] The hsaB gene is knocked out, namely ΔhsaB; the mutant strain in which the hsaB gene is knocked out is the ΔhsaB mutant strain. The hsaB gene is MyAD_24230; the ΔhsaB mutant strain is the ΔMyAD_24230 mutant strain.

[0221] In some preferred embodiments of the present disclosure, a method for constructing a recombinant bacterium for synthesizing progesterone as described above comprises the following steps: using genomic DNA as a template, amplifying the upstream and downstream sequences of the target sequence by PCR respectively; using conventional molecular biological methods to generate an integration plasmid containing a screening marker; for each disrupted gene, using conventional methods to transform the integration plasmid into a host bacterium, and culturing the host bacterium; after reverse screening, the recombinant bacterium is obtained. Preferably, the method obtains mutants in which the kasB gene is knocked out (ΔkasB), mutants in which the kstR gene is knocked out (ΔkstR), mutants in which the mam2A gene is knocked out (Δmam2A), mutants in which the lprL, mce2D, mce1C, mce1B and mce1A genes are knocked out (ΔMyAD_01575-01595), mutants in which the lprL gene is knocked out (ΔlprL), mutants in which the mce2D gene is knocked out (Δmce2D), mutants in which the mce1C gene is knocked out (Δmce1C), mutants in which the mce1B gene is knocked out (Δmce1B), mutants in which the mce1A gene is knocked out (Δmce1A), mutants in which the hsaADCB gene is knocked out (ΔhsaADCB), mutants in which the hsaA gene is knocked out (ΔhsaA), and mutants in which the hsaD gene is knocked out The invention relates to a mutant in which the hsaA gene is deleted (ΔhsaD), a mutant in which the hsaC gene is deleted (ΔhsaC), and a mutant in which the hsaB gene is deleted (ΔhsaB); the hsaADCB is a mixture of hsaA, hsaD, hsaC and hsaB genes; the lprL gene is MyAD_01575, the mce2D gene is MyAD_01580, the mce1C gene is MyAD_01585, the mce1B gene is MyAD_01590, and the mce1A gene is MyAD_01595; the lprL, mce2D, mce1C, mce1B and mce1A genes are represented by MyAD_01575-01595; the ΔMyAD_01575-01595 indicates that the lprL, mce2D, mce1C, mce1B and mce1A genes are deleted. More preferably, the method obtains mutants in which the kasB gene is knocked out (ΔkasB), mutants in which the kstR gene is knocked out (ΔkstR), mutants in which the mam2A gene is knocked out (Δmam2A), mutants in which the lprL, mce2D, mce1C, mce1B and mce1A genes are knocked out (ΔMyAD_01575-01595), mutants in which the hsaADCB gene is knocked out (ΔhsaADCB), and mutants in which the hsaA gene is knocked out (ΔhsaA). The hsaADCB is a mixture of hsaA, hsaD, hsaC and hsaB genes.The lprL gene is MyAD_01575, the mce2D gene is MyAD_01580, the mce1C gene is MyAD_01585, the mce1B gene is MyAD_01590, and the mce1A gene is MyAD_01595; the lprL, mce2D, mce1C, mce1B and mce1A genes are represented by MyAD_01575-01595; the ΔMyAD_01575-01595 indicates that the lprL, mce2D, mce1C, mce1B and mce1A genes are knocked out.

[0222] In some preferred embodiments of the present disclosure, a method for constructing a recombinant bacterium for synthesizing progesterone as described above comprises the following steps: using genomic DNA as a template, amplifying 800 to 1200 bp of the upstream and downstream sequences of the target sequence by PCR respectively; then inserting the amplified upstream and downstream homologous sequences into pJQ200SK using conventional molecular biological methods to generate an integration plasmid; the integration plasmid contains a gentamicin selection marker and a SacB counter-selection marker; for each disrupted gene, the corresponding integration plasmid is transformed into the host by electroporation using conventional methods. bacteria, and spread it on a medium containing gentamicin, and incubate it at 25-35°C for several days; inoculate a single colony into a 96-well plate of fresh pre-seed medium containing gentamicin, and incubate it in a shaker at 25-35°C for 1-6 days; for counter-screening, dilute the culture with sterile water and spread it on a medium, and incubate it at 25-35°C for 1-6 days; inoculate several colonies into fresh pre-seed medium in a 96-well plate, and incubate it in a shaker at 25-35°C for 1-6 days; the resulting culture is used as a template for PCR; after successful counter-screening, the recombinant bacteria are obtained. Preferably, the method obtains mutants in which the kasB gene is knocked out (ΔkasB), mutants in which the kstR gene is knocked out (ΔkstR), mutants in which the mam2A gene is knocked out (Δmam2A), mutants in which the lprL, mce2D, mce1C, mce1B and mce1A genes are knocked out (ΔMyAD_01575-01595), mutants in which the lprL gene is knocked out (ΔlprL), mutants in which the mce2D gene is knocked out (Δmce2D), mutants in which the mce1C gene is knocked out (Δmce1C), mutants in which the mce1B gene is knocked out (Δmce1B), mutants in which the mce1A gene is knocked out (Δmce1A), mutants in which the hsaADCB gene is knocked out (ΔhsaADCB), mutants in which the hsaA gene is knocked out (ΔhsaA), and mutants in which the hsaD gene is knocked out The invention relates to a mutant in which the hsaA gene is deleted (ΔhsaD), a mutant in which the hsaC gene is deleted (ΔhsaC), and a mutant in which the hsaB gene is deleted (ΔhsaB); the hsaADCB is a mixture of hsaA, hsaD, hsaC and hsaB genes; the lprL gene is MyAD_01575, the mce2D gene is MyAD_01580, the mce1C gene is MyAD_01585, the mce1B gene is MyAD_01590, and the mce1A gene is MyAD_01595; the lprL, mce2D, mce1C, mce1B and mce1A genes are represented by MyAD_01575-01595; the ΔMyAD_01575-01595 indicates that the lprL, mce2D, mce1C, mce1B and mce1A genes are deleted.More preferably, the method obtains mutants in which the kasB gene is knocked out (ΔkasB), mutants in which the kstR gene is knocked out (ΔkstR), mutants in which the mam2A gene is knocked out (Δmam2A), mutants in which the lprL, mce2D, mce1C, mce1B and mce1A genes are knocked out (ΔMyAD_01575-01595), mutants in which the hsaADCB gene is knocked out (ΔhsaADCB), and mutants in which the hsaA gene is knocked out (ΔhsaA). The hsaADCB is a mixture of hsaA, hsaD, hsaC and hsaB genes. The lprL gene is MyAD_01575, the mce2D gene is MyAD_01580, the mce1C gene is MyAD_01585, the mce1B gene is MyAD_01590, and the mce1A gene is MyAD_01595; the lprL, mce2D, mce1C, mce1B and mce1A genes are represented by MyAD_01575-01595; the ΔMyAD_01575-01595 indicates that the lprL, mce2D, mce1C, mce1B and mce1A genes are knocked out.

[0223] In some preferred embodiments of the present disclosure, the construction method of the mutant of Mycobacterium neoaurum is as follows: an unmarked gene deletion is performed in Mycobacterium neoaurum using an integration plasmid that cannot replicate in the bacteria. Mycobacterium neoaurum can be purchased, for example, through the website [Welcome to the ARS Culture Collection (NRRL) | ARS Culture Collection (usda.gov)].

[0224] In some preferred embodiments of the present disclosure, the construction method of the progestogen-producing bacteria mutant derived from the new golden Mycobacterium NRRL B-3805 is as follows: an unmarked gene deletion is performed in the new golden Mycobacterium using an integration plasmid that cannot replicate in bacteria. The progestogen-producing bacteria derived from the new golden Mycobacterium NRRL B-3805 contain the genetic modifications described in the previous patent application WO2024131933A1.

[0225] In some preferred embodiments of the present disclosure, the construction method of the mutant of Mycobacterium neoaurum or the mutant of progesterone-producing bacteria derived from Mycobacterium neoaurum NRRL B-3805 is as follows: using genomic DNA as a template, PCR is used to amplify the upstream and downstream 800 to 1200bp sequences of the target sequence respectively. Then, conventional molecular biological methods are used to insert the amplified upstream and downstream homologous sequences into pJQ200SK to generate an integrated plasmid. The integrated plasmid contains a gentamicin selection marker and a SacB reverse selection marker. For each gene deletion, the corresponding integrated plasmid is transformed into the host bacteria by electroporation using conventional methods, and is spread on a solid agar medium containing gentamicin and incubated at 28 to 32°C for several days. A single colony is inoculated into a 96-well plate containing a fresh pre-seed medium of gentamicin and incubated in a 28 to 32°C shaker for 2.5 to 3.5 days. For counterscreening, the culture is diluted with sterile water and plated on solid agar medium containing sucrose and incubated at 28-32°C for 3.5-4.5 days. Several colonies are inoculated into fresh pre-seed medium in a 96-well plate and incubated in a shaker at 28-32°C for 2.5-3.5 days. The resulting cultures are used as templates for PCR to determine whether they contain the desired marker-free gene deletion. After a successful counterscreen, additional gene deletions can be generated in a new host strain following the same procedure.Preferably, the method obtains mutants in which the kasB gene is knocked out (ΔkasB), mutants in which the kstR gene is knocked out (ΔkstR), mutants in which the mam2A gene is knocked out (Δmam2A), mutants in which the lprL, mce2D, mce1C, mce1B and mce1A genes are knocked out (ΔMyAD_01575-01595), mutants in which the lprL gene is knocked out (ΔlprL), mutants in which the mce2D gene is knocked out (Δmce2D), mutants in which the mce1C gene is knocked out (Δmce1C), mutants in which the mce1B gene is knocked out (Δmce1B), mutants in which the mce1A gene is knocked out (Δmce1A), mutants in which the hsaADCB gene is knocked out (ΔhsaADCB), mutants in which the hsaA gene is knocked out (ΔhsaA), and mutants in which the hsaD gene is knocked out The invention relates to a mutant in which the hsaA gene is deleted (ΔhsaD), a mutant in which the hsaC gene is deleted (ΔhsaC), and a mutant in which the hsaB gene is deleted (ΔhsaB); the hsaADCB is a mixture of hsaA, hsaD, hsaC and hsaB genes; the lprL gene is MyAD_01575, the mce2D gene is MyAD_01580, the mce1C gene is MyAD_01585, the mce1B gene is MyAD_01590, and the mce1A gene is MyAD_01595; the lprL, mce2D, mce1C, mce1B and mce1A genes are represented by MyAD_01575-01595; the ΔMyAD_01575-01595 indicates that the lprL, mce2D, mce1C, mce1B and mce1A genes are deleted. More preferably, the method obtains mutants in which the kasB gene is knocked out (ΔkasB), mutants in which the kstR gene is knocked out (ΔkstR), mutants in which the mam2A gene is knocked out (Δmam2A), mutants in which the lprL, mce2D, mce1C, mce1B and mce1A genes are knocked out (ΔMyAD_01575-01595), mutants in which the hsaADCB gene is knocked out (ΔhsaADCB), and mutants in which the hsaA gene is knocked out (ΔhsaA). The hsaADCB is a mixture of hsaA, hsaD, hsaC and hsaB genes.The lprL gene is MyAD_01575, the mce2D gene is MyAD_01580, the mce1C gene is MyAD_01585, the mce1B gene is MyAD_01590, and the mce1A gene is MyAD_01595; the lprL, mce2D, mce1C, mce1B and mce1A genes are represented by MyAD_01575-01595; the ΔMyAD_01575-01595 indicates that the lprL, mce2D, mce1C, mce1B and mce1A genes are knocked out. The progesterone-producing bacteria derived from the new golden Mycobacterium NRRL B-3805 include the genetic modifications described in the previous patent application WO2024131933A1.

[0226] A third aspect of the present disclosure provides use of the aforementioned recombinant bacteria or the aforementioned method in synthesizing progesterone.

[0227] The fourth aspect of the present disclosure provides a method for synthesizing progesterone, the method comprising: inoculating the recombinant bacteria as described above into a seed culture medium and mixing and incubating with a plant sterol emulsion to obtain progesterone; or, constructing a recombinant bacterium according to the method as described above, and inoculating the obtained recombinant bacteria into a seed culture medium and mixing and incubating with a plant sterol emulsion to obtain progesterone; the plant sterol emulsion is a plant sterol emulsion containing castor oil, water and stearic acid glyceryl. The method for synthesizing progesterone can increase the yield of progesterone, thereby reducing production costs. At the same time, the biosynthetic method for synthesizing progesterone removes the step of chemical synthesis, reduces the use of chemical solvents and greatly reduces environmental pollution, and solves the problem of serious chemical solvent pollution in the traditional process route.

[0228] In some preferred embodiments of the present disclosure, the incubation conditions include: an incubation temperature of 20 to 35°C, an incubation time of 1 to 5 days; the phytosterol emulsion is a 1 to 10% phytosterol emulsion comprising castor oil, water and glyceryl stearate. The incubation temperature is preferably 25 to 35°C, more preferably 28 to 32°C, and more preferably 29 to 31°C. The incubation time is preferably 1 to 4 days, more preferably 2 to 4 days, and more preferably 2.5 to 3.5 days. The phytosterol emulsion is preferably a 1 to 8% phytosterol emulsion comprising castor oil, water and glyceryl stearate; the phytosterol emulsion is more preferably a 1 to 5% phytosterol emulsion comprising castor oil, water and glyceryl stearate; the phytosterol emulsion is more preferably a 2 to 4% phytosterol emulsion comprising castor oil, water and glyceryl stearate.

[0229] In some preferred embodiments of the present disclosure, a method for synthesizing progesterone comprises: inoculating the recombinant bacteria as described above into a seed culture medium, mixing and incubating with a phytosterol emulsion to obtain progesterone; or, constructing a recombinant bacterium according to the method as described above, and inoculating the obtained recombinant bacteria into a seed culture medium, mixing and incubating with a phytosterol emulsion to obtain progesterone; preferably, the phytosterol emulsion is a phytosterol emulsion comprising castor oil, water and stearic acid glyceryl; preferably, the incubation conditions include: an incubation temperature of 20 to 35° C., and an incubation time of 1 to 5 days; preferably, the phytosterol emulsion is a 1 to 10% phytosterol emulsion comprising castor oil, water and stearic acid glyceryl.

[0230] In some preferred embodiments of the present disclosure, the phytosterol is one or a combination of β-sitosterol, campesterol, and cholesterol.

[0231] In some preferred embodiments of the present disclosure, the phytosterol is β-sitosterol.

[0232] In some more preferred embodiments of the present disclosure, the incubation conditions include: an incubation temperature of 25-35° C., and an incubation time of 2-4 days; and the phytosterol is a 1-5% phytosterol emulsion comprising castor oil, water and glyceryl stearate.

[0233] In some more preferred embodiments of the present disclosure, the incubation conditions include: an incubation temperature of 28-32° C., and an incubation time of 2.5-3.5 days; and the phytosterol is a 2-4% phytosterol emulsion comprising castor oil, water and glyceryl stearate.

[0234] In some preferred embodiments of the present disclosure, the method for synthesizing progesterone is as follows: the production of progesterone is increased by disrupting the kasB gene.

[0235] In some specific embodiments of the present disclosure, the method for synthesizing progesterone is as follows: kasB is knocked out in a progesterone-producing bacterium derived from the new golden mycobacterium NRRL B-3805 to obtain a ΔkasB mutant. 4 to 6 colonies of each bacterium (ΔkasB mutant, control bacterium) are inoculated into the wells of a 96-well plate containing a fresh pre-seed medium. The 96-well plate is incubated in a 28-32°C shaker for 2.5 to 3.5 days. The resulting culture is subcultured into a new 96-well plate containing a fresh seed medium, and the resulting 96-well plate is incubated in a 28-32°C shaker for 0.5 to 1.5 days. The resulting culture is transferred to a new 96-well plate, and 25 to 35 μL of a 2 to 4% phytosterol emulsion containing castor oil, water and stearyl glyceryl is added to each well. The resulting 96-well plate is incubated in a 28-32°C shaker for 1.5 to 2.5 days. After incubation, ethyl acetate was added to the 96-well plate, vortexed and shaken to extract the product. The 96-well plate was centrifuged, the organic layer was transferred to a new 96-well round-bottom plate, and further diluted before analysis by gas chromatography. The ΔkasB mutant can achieve gene disruption by markerless gene deletion as described above. The control bacteria are progesterone-producing bacteria derived from the new golden Mycobacterium NRRL B-3805 without corresponding genetic modifications. In this embodiment, the mutant bacteria in which kasB is knocked out (ΔkasB) produce more progesterone than the control bacteria after incubation with phytosterols for 2 to 3 days. It can be seen that the use of ΔkasB mutants can increase progesterone production, thereby reducing production costs. The progesterone-producing bacteria derived from the new golden Mycobacterium NRRL B-3805 contain genetic modifications described in the previous patent application WO2024131933A1.

[0236] In some preferred embodiments of the present disclosure, the method of synthesizing progesterone is as follows: the production of progesterone is increased by disrupting genes in the kstR and mce1 operons.

[0237] In some specific embodiments of the present disclosure, the method for synthesizing progesterone is as follows: in the progesterone-producing bacteria derived from the new golden mycobacterium NRRL B-3805, the genes in the kstR, mce1 operon and other genes are destroyed to obtain ΔkstR (ΔMyAD_24265) mutant bacteria, ΔMyAD_01560 mutant bacteria and ΔMyAD_01575-01595 mutant bacteria. 4 to 6 colonies of each bacterium (ΔMyAD_24265 mutant bacteria, ΔMyAD_01560 mutant bacteria and ΔMyAD_01575-01595 mutant bacteria, control bacteria) are inoculated into the wells of a 96-well plate containing a fresh pre-seed medium. The 96-well plate is incubated in a shaker at 28 to 32°C for 2.5 to 3.5 days. The resulting culture is subcultured into a new 96-well plate containing a fresh seed medium. The resulting 96-well plate is incubated in a shaker at 28 to 32°C for 0.5 to 1.5 days. The resulting culture was transferred to a new 96-well plate. 25 to 35 μL of a 2 to 4% phytosterol emulsion containing castor oil, water and stearin was added to each well. The resulting 96-well plate was incubated in a shaker at 28 to 32°C for 2.5 to 3.5 days. After incubation, ethyl acetate was added to the 96-well plate, vortexed and shaken to extract the product. The 96-well plate was centrifuged, the organic layer was transferred to a new 96-well round-bottom plate, and further diluted before analysis with RapidFire (Agilent). Gene disruption in the bacteria was confirmed by colony PCR. ΔkstR (ΔMyAD_24265) mutants, ΔMyAD_01560 mutants, and ΔMyAD_01575-01595 mutants can achieve gene disruption by markerless gene deletion as described above. The control bacteria were progesterone-producing bacteria derived from the new golden Mycobacterium NRRL B-3805 without corresponding genetic modification. In this embodiment, the above-mentioned ΔkstR (ΔMyAD_24265) mutant, ΔMyAD_01560 mutant and ΔMyAD_01575-01595 mutant are used to synthesize progesterone, which can significantly increase the production of progesterone compared with the control bacteria. The ΔkstR (ΔMyAD_24265) mutant has the most significant effect, and the yield is increased by 1.5 to 2.5 times compared with the control bacteria. Preferably, the ΔkstR (ΔMyAD_24265) mutant has the most significant effect, and the progesterone yield is increased by almost 2 times compared with the control bacteria. It can be seen that the use of ΔkstR (ΔMyAD_24265) mutant, ΔMyAD_01560 mutant and ΔMyAD_01575-01595 mutant can increase the production of progesterone, thereby reducing production costs. The new Mycobacterium aureum NRRL B-3805-derived progestogen contains genetic modifications described in the previous patent application WO2024131933A1.

[0238] In some preferred embodiments of the present disclosure, the method of synthesizing progesterone is as follows: progesterone production is increased by destroying hsaADCB.

[0239] In some specific embodiments of the present disclosure, the method for synthesizing progesterone is as follows: hsaA and hsaADCB are destroyed in the progesterone-producing bacteria derived from the new golden mycobacterium NRRL B-3805 to obtain ΔhsaA mutant bacteria and ΔhsaADCB mutant bacteria. 4 to 6 colonies of each bacterium (ΔhsaA mutant bacteria, ΔhsaADCB mutant bacteria, control bacteria) are inoculated into the wells of a 96-well plate containing fresh pre-seed culture medium. The resulting 96-well plate is incubated in a shaker at 28 to 32°C for 2.5 to 3.5 days. The resulting culture is passaged into a new 96-well plate containing fresh seed culture medium. The resulting 96-well plate is incubated in a shaker at 30°C for 0.5 to 1.5 days. The resulting culture is transferred to a new 96-well plate containing 15 to 25 μL of sunflower oil, soybean lecithin and phytosterols per well. The final concentrations of soy lecithin and phytosterols are 0.8-1.2 g / L and 8-12 g / L, respectively. The resulting 96-well plate was incubated in a shaker at 28-32° C. for 2.5-3.5 days. After incubation, ethyl acetate was added to the 96-well plate and vortexed to extract the product. The 96-well plate was centrifuged, and the organic layer was transferred to a new 96-well round-bottom plate and further diluted before analysis with RapidFire (Agilent). Gene disruption in the bacteria was confirmed by colony PCR. ΔhsaA mutants and ΔhsaADCB mutants can achieve gene disruption by markerless gene deletion as described above. The control bacteria are progesterone-producing bacteria derived from the new golden mycobacterium NRRL B-3805 without corresponding genetic modification. In this embodiment, the mutant bacteria with hsaADCB knockout (ΔhsaADCB) and the mutant bacteria with hsaA knockout (ΔhsaA) have significantly increased progesterone production compared to the control bacteria (Control). In addition, the effect of ΔhsaADCB is greater than that of ΔhsaA, that is, the progesterone production obtained by the ΔhsaADCB mutant is greater than that obtained by the ΔhsaA mutant. It can be seen that the use of ΔhsaA mutants and ΔhsaADCB mutants can increase progesterone production, thereby reducing production costs. The progesterone-producing bacteria derived from the new golden Mycobacterium NRRL B-3805 contain genetic modifications described in the previous patent application WO2024131933A1.

[0240] Example

[0241] In order to make the purpose, technical solution and advantages of the embodiments of the present disclosure clearer, the technical solution in the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, rather than all the embodiments. The following is only a further description of the present disclosure, and the protection scope of the present disclosure is not limited thereto.

[0242] Mycobacterium novoaureum NRRL B-3805 was purchased from the website: Welcome to the ARS Culture Collection (NRRL) | ARS Culture Collection (usda.gov).

[0243] Example 1

[0244] Preparation of progesterone-producing bacteria derived from Mycobacterium aureum NRRL B-3805:

[0245] In the same manner as Example 1 in WO2024131933A1, the new Mycobacterium aureum NRRL B-3805 was used to prepare the progesterone-producing bacteria derived from the new Mycobacterium aureum NRRL B-3805.

[0246] The progesterone-producing bacteria derived from the new Mycobacterium aureum NRRL B-3805 used in the following Examples 2, 3-1, 3-2, 4, 5, Comparative Example 1, 2, 3, and 4 are all progesterone-producing bacteria derived from the new Mycobacterium aureum NRRL B-3805 prepared in Example 1.

[0247] Example 2

[0248] An integrative plasmid that cannot replicate in bacteria was used to perform unmarked gene deletion in progestogens derived from new golden mycobacterium NRRL B-3805. The construction method of the progestogen mutant derived from new golden mycobacterium NRRL B-3805 is as follows: using genomic DNA as a template, PCR was used to amplify the upstream and downstream 800 to 1200bp sequences of the target sequence respectively. Then, conventional molecular biological methods were used to insert the amplified upstream and downstream homologous sequences into pJQ200SK to generate an integrated plasmid. The integrated plasmid contains a gentamicin selection marker and a SacB reverse selection marker. For each gene deletion, the corresponding integrated plasmid was transformed into the host bacteria by electroporation using conventional methods, and it was spread on a solid agar medium containing gentamicin and incubated at 30°C for several days. A single colony was inoculated into a 96-well plate containing fresh pre-seed medium with gentamicin and incubated in a 30°C shaker for three days. For counter-screening, the culture was diluted with sterile water and plated on solid agar medium containing sucrose and incubated at 30°C for four days. Several colonies were inoculated into fresh pre-seed medium in a 96-well plate and incubated in a 30°C shaker for three days. The resulting cultures were used as templates for PCR to determine whether they contained the desired marker-free gene deletions. After a successful counter-screening, other gene deletions can be generated in new host bacteria according to the same procedure. This method obtained mutants in which kasB was knocked out (ΔkasB), mutants in which kstR was knocked out (ΔkstR), mutants in which mam2A was knocked out (Δmam2A), mutants in which lprL, mce2D, mce1C, mce1B and mce1A genes were knocked out (ΔMyAD_01575-01595), mutants in which MyAD_01575 was knocked out (ΔMyAD_01575), and mutants in which MyAD_01575 was knocked out (ΔMyAD_01575) were obtained. 1580 knocked out (ΔMyAD_01580) mutant, MyAD_01585 knocked out (ΔMyAD_01585) mutant, MyAD_01590 knocked out (ΔMyAD_01590) mutant, MyAD_01595 knocked out (ΔMyAD_01595) mutant, hsaADCB knocked out (ΔhsaADCB) mutant, hsaA knocked out (ΔhsaA) mutant.

[0249] Example 3-1

[0250] Example of increasing progesterone production by knocking out kasB:

[0251] The ΔkasB mutant was obtained by knocking out kasB in the progesterone-producing bacteria derived from the new golden mycobacterium NRRL B-3805. The ΔkasB mutant was gene-disrupted by markerless gene deletion as described in Example 2. Six colonies of the ΔkasB bacteria were inoculated into the wells of a 96-well plate containing fresh pre-seed medium. The 96-well plate was incubated in a 30°C shaker for 3 days. The resulting culture was passaged into a new 96-well plate containing fresh seed medium, and the resulting 96-well plate was incubated in a 30°C shaker for 1 day. The resulting culture was transferred to a new 96-well plate, and 30 μL of a 3% phytosterol emulsion containing castor oil, water and stearyl glyceryl was added to each well. The resulting 96-well plate was incubated in a 30°C shaker for 2 days. After incubation, ethyl acetate was added to the 96-well plate, vortexed and shaken to extract the product. The 96-well plate was centrifuged and the organic layer was transferred to a new 96-well round-bottom plate and further diluted before analysis by gas chromatography. Figure 1 .

[0252] Comparative Example 1

[0253] Six colonies of the control bacteria (progesterone-producing bacteria derived from the new Mycobacterium aureum NRRL B-3805 without corresponding genetic modifications) were inoculated into the wells of a 96-well plate containing fresh pre-seed medium. The 96-well plate was incubated in a 30°C shaker for 3 days. The resulting culture was subcultured into a new 96-well plate containing fresh seed medium, and the resulting 96-well plate was incubated in a 30°C shaker for 1 day. The resulting culture was transferred to a new 96-well plate, and 30 μL of a 3% phytosterol emulsion containing castor oil, water and stearyl glyceryl was added to each well. The resulting 96-well plate was incubated in a 30°C shaker for 2 days. After incubation, ethyl acetate was added to the 96-well plate, vortexed and shaken to extract the product. The 96-well plate was centrifuged, and the organic layer was transferred to a new 96-well round-bottom plate and further diluted before analysis by gas chromatography. The progesterone production obtained by the control bacteria after 2 days of incubation with phytosterols is specifically described in detail. Figure 1 .

[0254] See also Figure 1 By comparing Example 3-1 with Comparative Example 1, it can be seen that the progesterone yield obtained in Example 3-1 is significantly increased. It is found that the above-mentioned ΔkasB mutant bacteria can significantly increase the progesterone yield in the process of synthesizing progesterone.

[0255] Example 3-2

[0256] Example of increasing progesterone production by disrupting kasB:

[0257] The ΔkasB mutant was obtained by knocking out kasB in a progestogen derived from the new golden mycobacterium NRRL B-3805. The ΔkasB mutant was gene-destroyed by markerless gene deletion as described in Example 2. Six colonies of the ΔkasB mutant were inoculated into the wells of a 96-well plate containing fresh pre-seed medium. The 96-well plate was incubated in a 30°C shaker for 3 days. The resulting culture was passaged into a new 96-well plate containing fresh seed medium, and the resulting 96-well plate was incubated in a 30°C shaker for 1 day. The resulting culture was transferred to a new 96-well plate, and 30 μL of a 3% phytosterol emulsion containing castor oil, water and stearyl glyceryl was added to each well. The resulting 96-well plate was incubated in a 30°C shaker for 3 days. After incubation, ethyl acetate was added to the 96-well plate, vortexed and shaken to extract the product. The 96-well plate was centrifuged and the organic layer was transferred to a new 96-well round-bottom plate and further diluted before analysis by gas chromatography. The ΔkasB mutants produced more progesterone than their isogenic strains after three days of incubation with phytosterols. Figure 1 .

[0258] Comparative Example 2

[0259] Six colonies of the control bacteria (progesterone-producing bacteria derived from the new Mycobacterium aureum NRRL B-3805 without corresponding genetic modification) were inoculated into the wells of a 96-well plate containing fresh pre-seed medium. The 96-well plate was incubated in a 30°C shaker for 3 days. The resulting culture was subcultured into a new 96-well plate containing fresh seed medium, and the resulting 96-well plate was incubated in a 30°C shaker for 1 day. The resulting culture was transferred to a new 96-well plate, and 30 μL of a 3% phytosterol emulsion containing castor oil, water and stearyl glyceryl was added to each well. The resulting 96-well plate was incubated in a 30°C shaker for 3 days. After incubation, ethyl acetate was added to the 96-well plate, vortexed and shaken to extract the product. The 96-well plate was centrifuged, and the organic layer was transferred to a new 96-well round-bottom plate and further diluted before analysis by gas chromatography. Progesterone production obtained by the control bacteria after 3 days of incubation with phytosterols (see for details) Figure 1 ).

[0260] See also Figure 1 By comparing Example 3-2 with Comparative Example 2, it can be seen that the progesterone yield obtained in Example 3-2 is significantly increased. The use of the above ΔkasB mutant bacteria to synthesize progesterone can significantly increase the yield of progesterone.

[0261] Example 4

[0262] Example of increasing progesterone production by disrupting genes in the kstR and mce1 operons:

[0263] In the progesterone-producing bacteria derived from the new golden mycobacterium NRRL B-3805, the genes in the kstR and mce1 operons and other genes were knocked out respectively to obtain ΔkstR (ΔMyAD_24265) mutants, ΔMyAD_01560 mutants and ΔMyAD_01575-01595 mutants. As described in Example 2, ΔMyAD_24265 mutants, ΔMyAD_01560 mutants and ΔMyAD_01575-01595 mutants were respectively gene-destroyed by marker-free gene deletion. Six colonies of each bacterium (ΔMyAD_24265 mutants, ΔMyAD_01560 mutants and ΔMyAD_01575-01595 mutants) were inoculated into the wells of a 96-well plate containing a fresh pre-seed culture medium. The 96-well plate was incubated in a 30°C shaking incubator for 3 days. The resulting culture was subcultured into a new 96-well plate containing fresh seed medium. The resulting 96-well plate was incubated in a 30°C shaker for 1 day. The resulting culture was transferred to a new 96-well plate. 30 μL of a 3% phytosterol emulsion containing castor oil, water and stearin was added to each well. The resulting 96-well plate was incubated in a 30°C shaker for 3 days. After incubation, ethyl acetate was added to the 96-well plate, vortexed and shaken to extract the product. The 96-well plate was centrifuged, and the organic layer was transferred to a new 96-well round-bottom plate and further diluted before analysis with RapidFire (Agilent). Gene disruption in the bacteria was confirmed by colony PCR. The progesterone production obtained by the ΔMyAD_24265 mutant, the ΔMyAD_01560 mutant and the ΔMyAD_01575-01595 mutant after 3 days of incubation with phytosterols is specifically described in detail. Figure 2 .

[0264] Comparative Example 3

[0265] Six colonies of the control bacteria (progesterone-producing bacteria derived from the new Mycobacterium aureum NRRL B-3805 without corresponding genetic modifications) were inoculated into the wells of a 96-well plate containing fresh pre-seed medium. The 96-well plate was incubated in a shaker at 30°C for 3 days. The resulting culture was subcultured into a new 96-well plate containing fresh seed medium. The resulting 96-well plate was incubated in a shaker at 30°C for 1 day. The resulting culture was transferred to a new 96-well plate. 30 μL of a 3% phytosterol emulsion containing castor oil, water and stearyl glyceryl was added to each well. The resulting 96-well plate was incubated in a shaker at 30°C for 3 days. After incubation, ethyl acetate was added to the 96-well plate, vortexed and shaken to extract the product. The 96-well plate was centrifuged, and the organic layer was transferred to a new 96-well round-bottom plate and further diluted before analysis with RapidFire (Agilent). The progesterone production obtained by the control bacteria after 3 days of incubation with phytosterols is specifically described in detail. Figure 2 .

[0266] See also Figure 2 By comparing Example 4 with Comparative Example 3, it can be seen that the progesterone yield obtained in Example 4 is significantly improved. The use of the above-mentioned ΔMyAD_24265 mutant, ΔMyAD_01560 mutant and ΔMyAD_01575-01595 mutant to synthesize progesterone can significantly increase the yield of progesterone. The strain with kstR knocked out (ΔMyAD_24265 mutant) has the most significant effect, and the yield is almost 2 times higher than that of the control strain.

[0267] Example 5

[0268] Example of increasing progesterone production by disrupting hsaA, hsaADCB:

[0269] In the progesterone-producing bacteria derived from the new golden mycobacterium NRRL B-3805, hsaA and hsaADCB were knocked out to obtain ΔhsaA mutants and ΔhsaADCB mutants. As described in Example 2, ΔhsaA mutants and ΔhsaADCB mutants achieve gene disruption by markerless gene deletion. Four colonies of each bacterium (ΔhsaA mutant, ΔhsaADCB mutant) were inoculated into the wells of a 96-well plate containing fresh pre-seed culture medium. The resulting 96-well plate was incubated in a 30°C shaker for 3 days. The resulting culture was passaged into a new 96-well plate containing fresh seed culture medium. The resulting 96-well plate was incubated in a 30°C shaker for 1 day. The resulting culture was transferred to a new 96-well plate containing 20 μL of sunflower oil, soybean lecithin and phytosterols in each well. The final concentrations of soybean lecithin and phytosterols were 1 g / L and 10 g / L, respectively. The resulting 96-well plate was incubated in a shaker at 30°C for 3 days. After incubation, ethyl acetate was added to the plate and vortexed to extract the product. The plate was centrifuged and the organic layer was transferred to a new 96-well round-bottom plate and further diluted before analysis with RapidFire (Agilent). Gene disruption in the bacteria was confirmed by colony PCR. The progesterone production obtained by the ΔhsaA mutant and the ΔhsaADCB mutant after 3 days of incubation with phytosterols is shown in Figure 3 The progesterone production of the ΔhsaADCB mutant after 3 days of incubation with phytosterols was significantly higher than that of the ΔhsaA mutant after 3 days of incubation with phytosterols, indicating that the influence of the ΔhsaADCB mutant was greater than that of the ΔhsaA mutant (see Figure 3 ).

[0270] Comparative Example 4

[0271] Four colonies of the control bacteria (progestogens derived from the new golden mycobacterium NRRL B-3805 without corresponding genetic modification) were inoculated into the wells of a 96-well plate containing fresh pre-seed medium. The resulting 96-well plate was incubated in a 30°C shaker for 3 days. The resulting culture was passaged into a new 96-well plate containing fresh seed medium. The resulting 96-well plate was incubated in a 30°C shaker for 1 day. The resulting culture was transferred to a new 96-well plate containing 20 μL of sunflower oil, soy lecithin and phytosterols per well. The final concentrations of soy lecithin and phytosterols were 1 g / L and 10 g / L, respectively. The resulting 96-well plate was incubated in a 30°C shaker for 3 days. After incubation, ethyl acetate was added to the plate and vortexed to extract the product. The plate was centrifuged, and the organic layer was transferred to a new 96-well round-bottom plate and further diluted before analysis with RapidFire (Agilent). The progesterone production obtained by the control bacteria after 3 days of incubation with phytosterols is shown in Figure 3 .

[0272] See also Figure 3 By comparing Example 5 with Comparative Example 4, it can be seen that the progesterone yield obtained in Example 5 is significantly improved. The use of the above-mentioned ΔhsaADCB mutant bacteria and ΔhsaA mutant bacteria to synthesize progesterone can significantly increase the yield of progesterone.

[0273] The complete chromosome genome of Mycobacterium neogoldensis NRRL B-3805 National Center for Biotechnology Information (NCBI) information: CP011022 (https: / / www.ncbi.nlm.nih.gov / search / all / ?term=CP011022).

[0274] Gene sequence information:

[0275] SEQ ID NO: 1: kasB(MyAD_17955)

[0276]

[0277] SEQ ID NO:2:mam2A(MyAD_01560)

[0278] GtgacggcagtcctcgacgacgcggtcgagctggacccggcaccgcaggagcccgcggcatcatggggggcccgggcggccgcgttcggcatcgacgtcctgttcggtgtggtgctgatcgtgatcctggcgatgctggcctggaccgcaccgttcctcggcgcgctgtggtgggtttacacggcgctggcggtgctgaccgggttgatcctgatgggcaaccgatgggtgctgccggtgcgcaccggctggaccgcgggccgcgccgtgaccgggatccgggtgttccgtacgtccggcgaccagaacccgcccgacaccggcaggctgatggtccgcgatgtcgcccatctgctcgacaccgttccgctgttggcggggtggttgtggccgttactcgaccgtcgccgccgcaccttcgccgacctgctgctgcgcaccgaggtccgccgcgtgggcgagccggagcgcgatgtgcgcagaccggtggcgctcggcctgatcggcgtggcgctggtctgcgtcggcgcggcgaccgcgggctacctcgtggtgtaccgcggtgaccgggcgctggaggatgcccgcggtcagatcgccgctcagggcgcaggcatcgtggagaagatgctgagctacaaccccgataccgtggatcaggatttcgagcgcgcccgcgcgttggccgccgacagctaccgcccacagttgatcgcacagcaggacgcattgcgcaaggccccgctggtgtccaacgagtactggtcggtcaacagcgcggtcctggagtcgcctccgccgacgacgaccaccgcatcgatgttgctggccatgcagggccagcgcggcagcaatcccggtgcaatgcgattcatcaccgcgaccgtgcgggtcgatttcgtcaagtccggtgagcagtggcaggtgcagaacctgtcggtgctgacccggccccagatgcaggcgagcggcggatgaSEQIDNO:3:lprL(MyAD_01575)

[0279]

[0280] SEQ ID NO:4:mce2D(MyAD_01580)

[0281]

[0282] SEQ ID NO:5:mce1C(MyAD_01585)

[0283]

[0284] SEQ ID NO:6:mce1B(MyAD_01590)

[0285]

[0286] SEQ ID NO:7:mce1A(MyAD_01595)

[0287]

[0288] SEQ ID NO:8:kstR(MyAD_24265)

[0289] Atgtcatcgccagcacaacccggatcgggttctgattcacctcgacaggtgatggccgtggccgtgctgtccgaatccgaactcgggtccgaggcccagcgggaacgacgtaagcgcatcctggatgccaccctggccatcgcctccaagggcggctacgaggcggtccagatgcgtgcggtggccgaacgcgccgatgtcgcggtgggcaccctgtaccgctacttcccgtcgaaggtgcacctgttggtgtcggccctgggccgggagttcgagcgcatcgatgccaaatccgaccgtgccacgctcaccggcggaacaccctacgagcgcctcaacgtgatggtgggcaggctgaaccgctccatgcagcgcaatccgctgctcaccgaggccatgacccgcgccttcgtcttcgccgatgcctcggcggccggtgaggtcgaccatgtcgggaaactcatggactcgatgttcgcccgcgcgatgagcgaaggcgagcccaccgaggaccagtaccacatcgccagggtgatctccgatgtctggctgtccaatctgctggcctggctgacccgccgcgcctcggccaccgatgtcgccaagcgcctggatctcgcggtccggctcctgatcggggacggcgaacaacctaaggtgtga

[0290] SEQ ID NO:9:hsaB(MyAD_24230)

[0291] Atgaccgccgaagccatcgatccacgtgccttccggaatgtgctcgggcagttctgcaccggtgtcaccgtcatcaccaccacggccgatgacgtgcccatcggcttcgcctgccagtcgttcgcggcgttgtccctggacccaccgttggtgttgttctgtccgaccaaacagtccagggcctggcaggccatcgaggccagcggcaggttctgcgtgaacatgctgcacgagaatcagcaacacgtctccgcgcagttcggatccaaggctccggacaagtttgccgggatcgactggaccgcatcgccgttgggctcaccgatcatcgacggcagcttggctcatatcgactgcaccgtcgcctctgtgcatgacggcggcgaccacttcgtggtgttcggcgcggtgcactcgttgtcggagccaccgtcgctcaaaccgcggccgctgctgttctaccgcggccagtacaccgggatcgagccggacaagaacaccccggcccagtggcgtgacgacctggaggccttcctgaccaccacgacctcggatacctggttataa

[0292] SEQ ID NO:10:hsaC(MyAD_24235)

[0293] Atgagcatcaagtcgctggggtacatgcgcatcgaggcaaccgatgtggctgcgtggcgcgagttcgccctgaaggtgctcggcatggtcgagggtcaggggtccacccccggtgccctctatctgcggatggacgaggttgccgcgcgcctggtcatcgtgcccggtgagcacgaccggctgctgatctccggatgggaggttgccgacgcccctgcgctgcaggcgctgcgcgagacactgtccaaggccggcgtcgagtacgccgagggcacccgcgacgagcgtgccgagcgccgcgtcgagggattgatccggttcaacgatccggcaggcaacgtcctggaggccttccacggggcccagtacctgggccgtcggttcgtcagcccgtatggccacaagttcgtcaccggtgaacaggggctcgggcatgtggtactcacgtgtgacgacgatgccgcggcgcaggcgttctatcaggacgtgctgggcttccggttgcgcgactcgatgagcctgccgccccagctcgccggccggcccgccgacggcgacccggtatggctgcgcttctacggttgcaacccgcgccaccacgcgctggcattcatgccgatgcccaaccccaccggcatcgtgcacctgatggtcgaggtcgaggagtccgatgatgtcggactgtgcctggaccgggcgttgcgccgcaatgtgaagatgtcggcgaccctcggcaggcacatcaacgacaagatgctgtccttctacatcaagactcctggcgggttcgatatcgaattcggctgcgagggattggaagtcgaggaccagaactgggttgcgcgggagagcaccgcggtcagcctgtgggggcacgacttcagcgtcggcttcaagtag

[0294] SEQ ID NO:11:hsaD(MyAD_24240)

[0295] AtgactgcggtttccgagatcactttcgagtccacttcccggtacgccgatgtgcaggccggtgacctggccatgcggttgcattaccacgaggcgggcgacccgacgtcgcagaccatcgtgctgctgcacggcggcggcccgggtgcgtcgagttggtcgaacttcggccgcaacatcgccgtactcgccgagcactaccacgtgttggccatcgaccagcccggctacgggctgtcggacaagcacaccgagcatgagcagtacaaccgctacagcgccaaggcggtgcgcggcctgctcgacacgctggagatcacgggacgtgttccgctgctgggcaattcgctcggcggcggcaccgcggtgcgcttcgcgctggattacccggaccgcgcgggcaagctcatcctgatgggccccggcggattgtcggtgaacctgttcgcccccgatcccaccgagggcgtcaaggcgctggcgaagttcaacttcgagccgacccgcgagaacctcgaggccttcatcaggatcatggtcttcgaccagaagctggtgaccccggaactggtcgaggaacgcttcaagatcgccagcaccccggagtcactggcggccacccgagcgatgggaaagtcgttcgcgggtcccgatttcgagctcggcatgatgtggcgcgaggtgtacaagctgcgccaaccggtgctgctgatctggggccgcgaggaccgggtgaacccgctcgacggcgcgctggtcgcggtcaagcagatcccgcgcgtgcagctgcatgtcttcgggcagtgcgggcactgggcgcaggtcgagaagttcgacgaattcaacaagctcaccattgatttcctgggctagSEQ ID NO:12:hsaA(MyAD_24245)

[0296]

[0297] The foregoing description of specific exemplary embodiments of the present disclosure is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present disclosure to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present disclosure and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present disclosure and various different selections and changes. The scope of the present disclosure is intended to be defined by the claims and their equivalents.

Claims

1. A recombinant bacterium for synthesizing progesterone, characterized in that: The recombinant bacteria are obtained by genetically modifying the starting bacteria; Wherein, the starting bacterium is Mycobacterium neoaurum, the genetic modification is gene disruption, at least part of the genes in the recombinant bacterium are disrupted, and the disrupted genes are selected from one or a mixture of the following gene clusters: Gene cluster 1: Gene cluster involved in fatty acid synthase II system, used to synthesize creatine; Gene cluster 2: Gene cluster encoding the mammalian cell entry (MCE) system related to fatty acid and cholesterol transport; Gene cluster 3: Gene cluster involved in sterol metabolism; The recombinant bacteria is used for increasing the yield of progesterone in progesterone synthesis.

2. The recombinant bacterium according to claim 1, wherein The gene cluster one includes the kasB gene; The gene cluster 2 includes genes related to the mce1 (mammalian cell entry) operon; preferably, the gene cluster 2 includes one or more of mam2A, lprL, mce2D, mce1C, mce1B and mce1A genes; The gene cluster three includes one or more of the hsaA, hsaD, hsaC, hsaB and kstR genes.

3. The recombinant bacterium according to claim 1 or 2, wherein The kasB gene is MyAD_17955, which includes a nucleotide sequence as shown in SEQ ID NO: 1 or having more than 90% homology thereto.

4. The recombinant bacterium according to any one of claims 1 to 3, wherein The mam2A gene is MyAD_01560, which includes a nucleotide sequence as shown in SEQ ID NO: 2 or having more than 90% homology thereto; Preferably, the lprL gene is MyAD_01575, which comprises a nucleotide sequence as shown in SEQ ID NO: 3 or having more than 90% homology thereto; Preferably, the mce2D gene is MyAD_01580, which comprises a nucleotide sequence as shown in SEQ ID NO: 4 or having more than 90% homology thereto; Preferably, the mce1C gene is MyAD_01585, which comprises a nucleotide sequence as shown in SEQ ID NO: 5 or having more than 90% homology thereto; Preferably, the mce1B gene is MyAD_01590, which comprises a nucleotide sequence as shown in SEQ ID NO: 6 or having more than 90% homology thereto; Preferably, the mce1A gene is MyAD_01595, which comprises a nucleotide sequence as shown in SEQ ID NO: 7 or having more than 90% homology thereto.

5. The recombinant bacterium according to any one of claims 1 to 4, wherein The kstR gene is MyAD_24265, which includes a nucleotide sequence as shown in SEQ ID NO: 8 or having more than 90% homology thereto; Preferably, the hsaB gene is MyAD_24230, which comprises a nucleotide sequence as shown in SEQ ID NO: 9 or having more than 90% homology thereto; Preferably, the hsaC gene is MyAD_24235, which comprises a nucleotide sequence as shown in SEQ ID NO: 10 or having more than 90% homology thereto; Preferably, the hsaD gene is MyAD_24240, which comprises a nucleotide sequence as shown in SEQ ID NO: 11 or having more than 90% homology thereto; Preferably, the hsaA gene is MyAD_24245, which comprises a nucleotide sequence as shown in SEQ ID NO: 12 or having more than 90% homology thereto.

6. The recombinant bacterium according to any one of claims 1 to 5, wherein The disrupted gene includes the kasB gene; preferably, the kasB gene is MyAD_17955, which includes a nucleotide sequence as shown in SEQ ID NO: 1 or having more than 90% homology thereto; or, The disrupted gene includes a kstR gene; preferably, the kstR gene is MyAD_24265, which includes a nucleotide sequence as shown in SEQ ID NO: 8 or having more than 90% homology thereto; or, The disrupted gene includes mam2A; preferably, the mam2A gene is MyAD_01560, which includes a nucleotide sequence as shown in SEQ ID NO: 2 or having more than 90% homology thereto; or, The disrupted genes include lprL, mce2D, mce1C, mce1B and mce1A genes; preferably, the lprL, mce2D, mce1C, mce1B and mce1A genes include nucleotide sequences as shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or having more than 90% homology thereto; or, The disrupted gene includes hsaA; preferably, the hsaA gene is MyAD_24245, which includes a nucleotide sequence as shown in SEQ ID NO: 12 or having more than 90% homology thereto; or, The disrupted gene includes hsaADCB; the hsaADCB is a mixture of hsaA, hsaD, hsaC and hsaB genes; preferably, the hsaADCB gene includes a nucleotide sequence as shown in SEQ ID NO: 12, SEQ ID NO: 11, SEQ ID NO: 10, SEQ ID NO: 9 or a nucleotide sequence having more than 90% homology thereto.

7. The recombinant bacterium according to any one of claims 1 to 6, wherein The starting bacteria is new golden mycobacterium NRRLB-3805.

8. The recombinant bacterium according to any one of claims 1 to 7, wherein The gene disruption method is achieved by genetic engineering technology, resulting in a decrease in the level of the protein encoded by the disrupted gene; Preferably, the gene disruption method comprises random mutation of protein sequence, Rec / ET method, CRISPR-based genome editing or a combination thereof; Preferably, the gene disruption is by allelic exchange with an integration plasmid.

9. A method for constructing a recombinant bacterium for synthesizing progesterone according to any one of claims 1 to 8, characterized in that: The method comprises: genetically modifying a starting bacterium to obtain a recombinant bacterium according to any one of claims 1 to 8, so that the level of a protein encoded by a destroyed gene of the starting bacterium is reduced; The method in which the level of the protein encoded by the destroyed gene of the starting bacteria is reduced is called gene destruction.

10. The method according to claim 9, wherein: The gene disruption method is achieved by genetic engineering technology; Preferably, the gene disruption method comprises random mutation of protein sequence, Rec / ET method, CRISPR-based genome editing or a combination thereof; Preferably, the gene disruption is by allelic exchange with an integration plasmid.

11. The method according to claim 9 or 10, wherein: The method comprises the following steps: Genomic DNA was used as a template to amplify the upstream and downstream sequences of the target sequence by PCR; Conventional molecular biology methods are used to generate an integrating plasmid containing a selectable marker; For each disrupted gene, the integration plasmid was transformed into the host bacteria using conventional methods. Cultivating the host bacteria; After reverse screening, the recombinant bacteria are obtained.

12. Use of the recombinant bacteria according to any one of claims 1 to 8 or the method according to claim 9 or 10 in synthesizing progesterone.

13. A method for synthesizing progesterone, characterized in that: The method comprises: inoculating the recombinant bacteria according to any one of claims 1 to 8 into a seed culture medium, mixing and incubating with a plant sterol emulsion to obtain progesterone; Alternatively, constructing a recombinant bacterium according to the method of claim 9 or 10, and inoculating the obtained recombinant bacterium into a seed culture medium and mixing and incubating with a plant sterol emulsion to obtain progesterone; Preferably, the phytosterol emulsion is a phytosterol emulsion comprising castor oil, water and glyceryl stearate; Preferably, the incubation conditions include: an incubation temperature of 20 to 35° C. and an incubation time of 1 to 5 days; Preferably, the phytosterol emulsion is a 1-10% phytosterol emulsion comprising castor oil, water and glyceryl stearate.

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