3-ethanone deoxycholic acid compound as well as synthetase, preparation method and application of 3-ethanone deoxycholic acid compound

Abstract

The invention relates to the field of microbiology and biochemistry, in particular to a 3-ethanone deoxycholic acid compound, a synthetase thereof, a preparation method and application of the 3-ethanone deoxycholic acid compound. According to the present invention, the double-tailed acid compound represented by 3-ethanone deoxybile acid is found, the enzymatic experiment verifies that the ADS sequence can catalyze the generation of the double-tailed acid compound, the bacterial strain capable of generating the double-tailed acid compound is further screened through the experiment, and the growth promotion effect of 3-ethanone deoxycholic acid on lactic acid bacteria is found through detection; the method can be used for intervention of diseases.

Description

Technical Field

[0001] This invention relates to the fields of microbiology and biochemistry, specifically to 3-acetone deoxycholic acid compounds, their synthases, preparation methods, and uses. Background Technology

[0002] Steroids in the human body are a class of compounds crucial to physiological functions, including cholesterol, bile acids, glucocorticoids, and sex hormones. They play a wide range of roles in regulating growth and development, reproduction, immune responses, inflammation, metabolism, and nervous system function. The gut microbiota plays a vital role in the metabolism and transformation of these substances. Taking bile acids as an example, gut microbiota-mediated bile acid modifications currently include: 1) binding and unbinding; 2) redox-mediated stereoisomerization; 3) sulfonation; 4) 7-dehydroxylation; and 5) 3-acylation. However, all currently discovered bile acid modifications occur at active functional groups such as hydroxyl and carboxyl groups. Compared to common functional group modifications, reactions that alter the carbon-carbon skeleton of metabolites are relatively rare and limited in type, but such changes often significantly affect metabolite activity (for example, the microbial ring-opening metabolism of cholesterol can form a metabolite with a completely new skeleton, initially named calcitrated cholesterol; with the understanding of its unique physiological and pathophysiological functions, a completely new type of metabolite, namely vitamin D, has gradually emerged). It remains unclear whether bile acids with carbon-carbon skeletons exist.

[0003] None of the known modifications alter the carbon skeleton of these substances. There are no reports on whether gut bacteria mediate skeleton modifications in these substances. It is unclear whether potential new substances exist in the human population and what their functions are. Summary of the Invention

[0004] In order to overcome the shortcomings of the prior art and enrich the prior art, the present invention provides a series of novel 3-ethylhexanoate deoxycholic acid compounds, their synthases, producing bacteria and uses.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] The object of this invention (1) is to provide a series of novel 3-acetone deoxycholic acid compounds, which are called di-tailed acids. This invention provides the compound (3-acetone deoxycholic acid) shown in Formula I:

[0007]

[0008] Or the compound shown in Formula II:

[0009]

[0010] Or the compound shown in Formula III:

[0011]

[0012] Or the compound shown in Formula IV:

[0013]

[0014] Or the compound shown in formula V:

[0015]

[0016] Or the compound shown in Formula VI:

[0017]

[0018] Or the compound shown in formula VII:

[0019]

[0020] Or the compound shown in Formula VIII:

[0021]

[0022] The object (2) of this invention is to provide a synthase ADS1-12 for the synthesis of 3-ethylketone deoxycholic acid compounds shown in Formulas I-VIII, wherein the amino acid sequences of the synthase are shown in SEQ ID NO:1-12.

[0023] The compounds provided in this invention are all newly discovered and can be prepared by in vitro enzymatic reactions.

[0024] The objective (3) of this invention is to provide an in vitro enzymatic method for preparing 3-acetone deoxycholic acid compounds shown in formulas I-VIII, the specific preparation method of which is as follows:

[0025] 1) Obtain the catalytic enzyme ADS by overexpressing any one of ADS1-12 in Escherichia coli;

[0026] 2) The ADS protein was incubated with substrates 3-oxocholic acid (3-oxoCA), 3-oxodeoxycholic acid (3-oxoDCA), 3-oxochenodeoxycholic acid (3-oxoCDCA), 3-oxolithocholic acid (3-oxoLCA) and pyruvate at 37°C for 10-16 hours.

[0027] 3) The 3-acetone deoxycholic acid compounds were separated by HPLC-ELSD.

[0028] The objective (4) of this invention is to provide a method for preparing 3-acetone deoxycholic acid compounds as shown in formulas I-VIII by microbial fermentation, the specific method of which is as follows:

[0029] 1) Activated strains of Bacteroides (including Bacteroides fowleri, Bacteroides ehrlichii, Bacteroides fowleri, Bacteroides fowleri, Bacteroides fragilis, Bacteroides fowleri, Bacteroides koreanum, Bacteroides ovalis, Bacteroides polymorpha, Bacteroides xylana), Clostridium (including Clostridium neonatalis, Clostridium putrefactiveis), Enterococcus (including Enterococcus staphylococcus, Enterococcus faecalis, Enterococcus lactis), Escherichia (including Escherichia coli), Eubacterium (including Eubacterium indolentum), Trichophyton (including Trichophyton styloides), Lactobacillus (including Lactobacillus baltii), Lactobacillus reuteri), Lactobacillus (including Lactobacillus fermentum and Lactobacillus gasseri), Parabacterium (including Parabacterium difficile), Clostridium (including Clostridium sovoides), and Ruminococcus (including active Ruminococcus) were inoculated into fermentation seed culture media (Bacteroides, Clostridium, Enterococcus, Escherichia, Eubacterium, Trichophyton, Parabacterium, Clostridium, and Ruminococcus were inoculated into GAM medium at a ratio of 5%; Lactobacillus and Lactobacillus were inoculated into MRS medium at a ratio of 5%).

[0030] 2) Transfer the fermentation seed liquid to a fermentation medium containing substrates 3-oxoCA, 3-oxoDCA, 3-oxoCDCA, and 3-oxoLCA, and anaerobic ferment for 48 hours (the fermentation medium for Bacteroides, Clostridium, Enterococcus, Escherichia, Eubacterium, Trichophyton, Parabacterium, Paraclostridium, and Ruminococcus was GAM medium; the fermentation medium for Lactobacillus and Lactobacillus was MRS medium).

[0031] 3) Extract and concentrate the fermentation product, and separate the 3-acetone deoxycholic acid compounds using HPLC-ELSD.

[0032] Preferably, in objectives (3) and (4) of this invention, the HPLC-ELSD separation conditions are as follows: a 5C18-MS-II column (4.6 mm × 250 mm) is used, the column temperature is 35 °C, and the flow rate is 1.0 mL / min. Mobile phase A is a 0.1% formic acid aqueous solution, and mobile phase B is a 0.1% formic acid acetonitrile solution. Purification is carried out under isocratic elution conditions of mobile phase A:B = 60:40 (v / v).

[0033] The 3-ethylketone deoxycholic acid compounds shown in Formulas I-VIII provided by this invention can be synthesized by those skilled in the art through chemical methods, in addition to the above-mentioned enzymatic reactions and microbial fermentation culture.

[0034] The object (5) of the present invention is to provide the use of the 3-ethyl ketone deoxycholic acid compounds shown in Formulas I-VIII, which promote the growth of lactic acid bacteria for disease intervention.

[0035] Preferably, the lactic acid bacteria include, but are not limited to, *Lactobacillus gasseri*, *Lactobacillus helveticus*, *Lactobacillus curvatureii*, and *Lactobacillus johnsonii* strains.

[0036] Preferably, the disease is selected from metabolic-associated fatty liver disease (MAFLD).

[0037] Compared with the prior art, the present invention has the following advantages:

[0038] Through extensive and in-depth research and experiments, the inventors discovered di-tailed acid compounds, represented by 3-acetyl-deoxycholic acid. Enzymatic experiments verified that ADS-like sequences (such as ADS1-12) can catalyze the generation of di-tailed acid compounds. Further experiments screened strains that can produce di-tailed acid compounds and detected the growth-promoting effect of 3-acetyl-deoxycholic acid on lactic acid bacteria, which can be used for disease intervention. Attached Figure Description

[0039] Figure 1 : Chemical structure characterization spectrum of compound II.

[0040] Figure 2 : Chemical structure characterization spectrum of compound III.

[0041] Figure 3 : Chemical structure characterization spectrum of compound IV.

[0042] Figure 4 : Chemical structure characterization spectrum of compound V.

[0043] Figure 5 : Chemical structure characterization spectrum of compound VI.

[0044] Figure 6 : Chemical structure characterization spectrum of compound VII.

[0045] Figure 7 : Chemical structure characterization spectrum of compound VIII. Detailed Implementation

[0046] Through extensive and in-depth research and experimentation, the inventors discovered di-tailed acid compounds, represented by 3-acetylenedeoxycholic acid. Enzymatic experiments verified that ADS-like sequences (such as ADS1-12) can catalyze the formation of di-tailed acid compounds. Further experiments screened strains capable of producing di-tailed acid compounds, and the growth-promoting effect of 3-acetylenedeoxycholic acid on lactic acid bacteria was detected. Based on these findings, this invention was completed. Specific embodiments are described below, but the invention is not limited thereto.

[0047] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0048] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0049] Example 1: ADS activity assay of two-tailed acid synthase sequences

[0050] A class of ThDP-dependent enzymes that can utilize 3-oxoDCA was screened from the genome of intestinal bacteria and named ADS1-12. The amino acid sequences are shown in SEQ ID NO:1-12.

[0051] >ADS-1(SEQ ID NO:1)

[0052] MYLENIYSPADVKKLSFKELNDLSNEIRASLLQKLSEHGGHFGPNFGMVEATIALHYVFNSPEDKLVFDVSHQSYVHKMLTGRKDAFLYPAEYDNVSGYSEPQESKHDFFVIGHTSTVSLASGLAKGRDLTGGNENIIAVIGDGS LSGGEAFEGLDYVAELGTNMIIIVNDNQMSIAENHSGLYKNLKKLRDSNGQCECNFFKAMGLDYMYVNDGNNVEALVKAFSKVKDIQHPVVVHINTLKGKGYKPAEQDRETFHWLTPFNIETGKPKFAEDAEDYSEMTAQYLLKKM KEDKRVVTITSGTPAVLGFTPDRRKEAGKQFVDVGIAEEHAVALASGIAANGGKPVYGVYSTFIQRSYDQLSQDLCINNNPAVLLLVFWGTLSGMNDVTHLCFFDIPLISNIPNMVYLAPTCKEEYLAMLEWSIRQNEYPVAIRVPA TDVITCGEPVETDYSVLNRYNVTHRGAKVAILALGSFYGLGQSVASLLKEKANIDATLINPRYITGVDNELMDELKAADHELVITLEDGVLDGGFGEKIARYYGATNMKVLNFGAKKEFVDRYDIQEFLRANHLTDEQIVEDITAVIG

[0053] >ADS-2(SEQ ID NO:2)

[0054] MYIENINGPEDVKKLSEDQLNVLAEEIRDALLKKLSKHGGHFGPNFGMVEATIAMHYVFESPKDKIVYDVSHQSYPHKMLTGRKDAFLYEEHYDDVSGYSNPGESEHDHFTIGHTSTSISLALGLAKARDLKEENGNVIAVIGDGSLSGGEALEGLDYAAELGGNLIIVVNDNDMSIAENHGGLYENLKALRETNGESKCNLFKAMGLDYIYVKEGNKVADLIEAFKQVKDTKKAVVVHINTQKGKGYKLAEEHKEDWHYCAPFDVETGKPLDSFDGEDFSSVTAQYLLKKMKEDKKVVAITSATPTVMGFTEDKRKEAGKQFIDVGIAEETAMAMAAGIAAGGGKPVYGVYSTFVQRTYDQITQDLCIDSNAATIVTFWGSVYGMNDVTHLGLQDIPMMSNIPNLVYLAPTTKEEYLAMLDWSIDQDQYPVAIKLPGGEMISDGKKITKDFSQLNKYEVTEKGSKVAVIGLGTFYNMGCEVAKAVEEKTGTKATVINPYYITGIDEVLLEDLKKDHDVVITLEDGFLEGGFGEKIARFYGDSDMKVFNYGVKKELLDRYDIEELLKKNHLTVQQIVGDLKF

[0055] >ADS-3(SEQ ID NO:3)

[0056] MYLENIYSPADVKKLSFQELNDLSNEIRASLLQKLSEHGGHFGPNFGMVEATIALHYVFNSPEDKIVFDVSHQSYVHKMLTGRKDAFLYPAEYDNVSGYSEPHESKHDFFVIGHTSTSVSLASGLAKGRDLTGGNENIIAVIGDGS LSGGEAFEGLDYVAELGTNMIIIVNDNQMSIAENHGGLYKNLKDLRDSNGQCECNFFKAMGLDMYVNDGNNVEALVEAFSKVKDIQHPIVVHINTLKGKGYGRAELDKETYHWRTPFNIETGESKFGETAEDYSEVTAQYLLKKM KEDKRVVTITSGTPAVLGFTPDRRQEAGKQFVDVGIAEEHAVALASGIAANGKKPVYGVYSTFIQRSYDQLSQDLCINNPAVLLVFWGTLSGMNDVTHLCFFDIPLISNIPNMVYLAPTCKEEYLAMLEWGIRQNEHPVAIRVPA TDVISCGESVESDYSVLNRYKVAHCGSKVAILALGSFFGLGQSVLSLLKEKAGIDATLINPRYITGIDNELMEELKADHQLVITLEDGVLDGGFGEKIARYYGTSDMKVLNYGAKKEFVDRYDIQELLRANHLTDELIVEDIRKIIG

[0057] >ADS-4(SEQ ID NO:4)

[0058] MYIEKINGPADVKRLSAGELETLSEEVRSVLLKKLSEHGGHVGPNLGMVEATIALHYVFNSPIDKIVYDVSHQSYTHKILTGRKAAFMNAAEYDEVSGYTEPSESEHDFFVIGHTSTSVSLATGLAKARDLKGDTGNVIAVIGDGSLSGGEAFEGLNNAAELGTNFIVIVNDNQMSIAENHGGLYRNLQLLRDTDGQAPCNFFTAMGLDYLYVKDGNDVQALISAFQKVKDIDHPVVHINTLKGKGYRLAEQQQERFHYSIPFSLETGNPSVDFGNEEDYGDLTARYLLQEM KRDRTIVGITAGTPTVFGFTAERREEAGRQFVDVGIAEEHAVAMASAIAAGGGKPVFGVYSTFIQRAYDQLSQDLCINNNPALILVFWGGASSMNDVTHLCLFDIPVIGNIPNMVYLAPTCREEYLAMLEWGIHQTKHPVAVRVPA NGVISRGIEPEKDYGKLLNRYEVSRRGEKVAVLGLGSFYQLGEEVVRKLKEDTGIDATLVNPRFITGVDEELLEDLKCDHTLVVTLEDGVLDGGFGEKIARYYASSDMKVLNYGIKEFADRYDISELLRDNRLTASQIADDIKRVR

[0059] >ADS-5(SEQ ID NO:5)

[0060] MYLENIYSPEDVKKLSIEELNKLSGEIRAALLQKLSEHGGHFGPNFGMVEATIALHYVFNSPKDKIVFDVSHQSYVHKMLTGRKDAFLNPAKYDNVSGYTEPQESEHDFFIIGHTSTSVSLASGLAKGRDLTGGNENIIAVIGDGS LSGGEAFEGLDYVAELGTNMIIIVNDNQMSIAENHGGLYKNLKELRDSNGQCECNFFKAMGLDYMYVNDGNNVQALIEFSKIKDIQHPIVVHINTLKKGGYARAEQDKETYHWRTPFNPKTGEAKVNDEEEDYSEVTAQYLLQKM KEDPRVVTITSGTPAVLGFTPDRRQEAGKQFVDVGIAEEHAVALASGIAANGGKPVYGVYSTFIQRSYDQLSQDLCINNNPAVLLVFWGTLSGMNDVTHLCFFDIPVISNIPNMVYLAPTCKEEYIAMLEWSIRQNEHPVAIRVPATEVISCGEPVDSDYSILNRYKVTHRGSKVAIVALGSFYGLGQSVASLLKEKENIDATLINPRYITGIDSELMNELKADHELVITLEDGVLDGGFGEKISRYYGTSDMKVLNYGAKKEFVDRYDLQEFLRVNHLTDEQIVEDITALIG

[0061] >ADS-6(SEQ ID NO:6)

[0062] MYIEKINGPADVKRLSAGELETLSEEVRGVLLKKLSEHGGHVGPNLGMVEATVALHYVFNSPADKIVYDVSHQSYAHKILTGRKAAFMDAAAYDEVSGYTEPSESEHDFFVIGHTSTSVSLATGLAKARDLKGGTGNVIAVIGDGSLSGGEAFEGLNNAAELGTNFIVIVNDNQMSIAENHGGLYRNLQLLRETDGQAPCNFFTAMGLDYLYVKDGNNVQALIDAFRKVKDIAHPVAVHINTLKGKGYRLAEQQQERFHYSAPFCLETGSPAVDSGNEEDYGDLTARYLLQEM KKDRTVVGITAGTPTVFGFTPPERREEAGRQFVDVGIAEEHAVAMASAIAAGGGKPVFGVYSTFIQRAYDQLSQDLCINNPALILVFWGGASTMNDVTHLCLFDIPVIGNIPNMVYLAPTCREEYFAMLEWGIHQTEHPVAIRVPA NGVLSRGIEPEKDYGKLLNRYEVAYRGGQVAVLGLGSFFQLGEEVVGKLKEETGVDATLVNPRFITGVDEALLEDLKRDHTLVVTLEDGVLDGGFGEKIARYYGTSGMKVLNYGIRKEFADRYDAGELLRDNRLTVPQIVEDIKKVR

[0063] >ADS-7(SEQ ID NO:7)

[0064] MYLENIYSPADVKKLSFQELNDLSHEIRASLLQKLSAHGGHFGPNFGMVEATIALHYVFNSPKDKIVYDVSHQSYVHKMLTGRKDAFLHPAEYDHVSGYSEPQESEHDFFVIGHTSTSVSLASGLAKGRDLTGGNENIIAVIGDGSLSGGEAFEGLDYVAELGTNMIIIVNDNQMSIAENHGGLYKNLKDLRDSNGQCECNFFKAMGLDYMYVNDGNCVEALIEAFSKVKDIQHPIVVHINTLKKGYEPAEQDKETYHWRTPFDLETGKSKMNDDAEDYSEVTAQYLLKKM KEDKRVVTITSGTPAVLGFTPDRRQEAGKQFVDVGIAEEHAVALASGIAANGGKPVYGVYSTFIQRSYDQLSQDLCINNNPAVLLVFWGTLSGMNDVTHLCFFDIPLISNIPNMVYLAPTCKEEYLAMLEWSIHQNEHPVAIRVPATDVISCGEPVESDYSNLNRYKVAHRGSKVAILALGSFFGLGQSVLSLLKEKANIDATLINPRYITGVDSELMDELKADHELVITLEDGVLDGGFGEKIARYYGATDMKVLNYGAKKEFVDRYDIQELLRANHLTDEQIVEDILSLIG

[0065] >ADS-8(SEQ ID NO:8)

[0066] MYIEKVNSPADVKKLSVSEMNGLGGEIRQVLLNKLSSHGGHVGPNLGMVEATIALHYVFDSPTDKIVYDVSHQSYTHKILTGRRAAFMNADEYDEVSGYSEPSESEHDFFVIGHTSTSVSLASGLAKARDLKGEAGNVIAVIGDGSLSGGEAFEGLNVGAELGTNVIVIVNDNQMSIAENHGGLYRNLQQLRETEGQAPCNYFKAMGYDYLYVKDGNDVEQLIEAFREVKDKKHPVVHINTLKGKGYKLAEEQKERFHYSVPFDLETGNLTGEPGKGEDYADLTAGYLLQEM KKDPTVVGITAGTPTVFGFTPERRKEAGRQFIDMGIAEEQAVAMASAIAAGGGKPVFGVFGVYSTFIQRAYDQLSQDLCINNNPALILVFWGSLSSMNDVTHLCLFDIPVIGNIPNMVYLAPTCREEYMAMLEWGIRQTEHPVAIRVPANGVVRRNVEPEKDYGKTLNRYEVSHRGEKKVAILGLGSFYQLGEAVAARLKEEKGVDATLVNPRYITGVDEALLDDLKLDHTLVITLEDGVLDGGFGEKIARYYGPSDMKVLNYGVKKEFIDRYDVEEQLKKNRLTVPQIVEDICRIC

[0067] >ADS-9(SEQ ID NO:9)

[0068] MYIENINGPQDVKKLTVDEMRTLADEMRHALLIRASKHGGHFGPNFGMVEATIALHYVFESPKDKIVYDVSHQSYPHKMLTGRKDAYLYEEHYDDVTGYSNPHESEHDFFTVGHTSTSISLAAGLAKARDLQGAEGNVIAVIGDGSLSGGEALEGLDYAKELDGNLIIVVNDNDMSIAENHGGLYTNLRLLRDTNGTAECNLFRAVGLDYYYVHEGNDVAALIDTFQKVKDSKKPVVVHIRTLKKGGYAPAEEHKEQWHYSGPFDIETGRSLFEGDEEDYSSVSCDYLLDK MKKDPKVVAITAGTPTVIGFTEDKRREAGKQFVDVGIAEETAVALASGIAAGGGKPVFGVYSSFVQRTFDQISQDLCINNNAATIVTFAGSVYGMNDVTHLGFQDIPMLSNIPNLVYLAPTTKEEYIAMLDWSIEQNEHPVAIKLPGGAMVSGSPVTKNFGDLNKYEVTQKGSKVALLGLGTFYSLANAAADEMKSELGIDATVINPYYITGLDEELLESLKADHSVVVTIEDGILDGGFGEKIARFYGDSDMKVLNYGLKKEFLDRYDVEEVLKDNRLDAEMIAEDVKGLL

[0069] >ADS-10(SEQ ID NO:10)

[0070] MYIEQVNSPQDIKRFSAEQLRQLAGEVRNALLTKLSAHGGHVGPNLGMVEATIALHYVFNSPTDKMVYDVSHQSYTHKMLTGRKDAFLNPEDYDVVSGYTNPRESGHDFFTIGHTSTSVSLACGLAKARDLKGGHENIIAVIGDGSLSGGEAYEGLSNAGEMGTNLIIVVNDNEMSIAENHGGLYQNLKELRDTEGRSSCNFFRSLGLDYLYVGEGNDIPSLVAAFAKVKDTSRPTVVHIHTQKGKGYAPAEADREEFHWEMPFDLETGKPKVDCSGMEDYHSLTGKFLLEKMKEDHTVVAISSGTPTVIGFTPERRKQAGRQFVDVGIAEEHAVALASGIAAGGGRPVYGVYSTFVQRCYDQLSQDLCINGNPAVITVFMGTVAGMNDVTHLGFFDIPLISNIPNMVYLAPTCSEEYFAMLEWAVRQTEYPVAIRVPGAAVVHRKEEFDTDYSELNRYKMTARGETVAILALGAFYDLGQALKNKLREESGVEATLINPRYITGLDEPMLEELKVGHRIVVTLEDGVLDGGFGEKIARYYGNSEMRVLNYGLRKEFADRYNLDELMKANRLTDKQMAEDIAGILG

[0071] >ADS-11(SEQ ID NO:11)

[0072] MYIEQVNSPQDIKQFSAEQLRQLAGEVRNALLTKLSAHGGHVGPNLGMVEATIALHYVFNSPTDKMVYDVSHQSYTHKMLTGRKGAFLNPEDYDVVSGYTNPRESGHDFFTIGHTSTSVSLACGLAKARDLKGGHENIIAVIGDGSLSGGEAYEGLSNAGEMGTNLIIVVNDNEMSIAENHGGLYQNLKELRDTEGRSSCNFFRSLGLDYLYVGEGNDIPSLVAAFAKVKDTSRPTVVHIHTQKGKGYAPAEADREEFHWEMPFNLETGKPKVDCSGMEDYHSLTGKFLLEKMKEDHTVVAISSGTPTVIGFTPERRKQAGRQFVDVGIAEEHAVALASGIAAGGGRPVYGVYSTFIQRCYDQLSQDLCINGNPAVITVFMGTVAGMNDVTHLGFFDIPLISNIPNMVYLAPTCSEEYFAMLEWAVRQTEYPVAIRVPGVAVVHRKEEFDTDYSELNRYKMTARGETVAILALGSFYDLGQALKNKLREESGVEATLINPRYITGLDEPMLEELKVGHRIVVTLEDGVLDGGFGEKIARYYGNSEMRVLNYGLRKEFADRYNLDELMKANRLTDKQMAEDIAGILE

[0073] >ADS-12(SEQ ID NO:12)

[0074] MYLENIYSPADVKKLSEKELNELGGEIRAALLQKLSEHGGHFGPNFGMVEATIALHYVFNSPKDKIVFDVSHQSYVHKMLTGRKDAFLHPEKYDNVSGYTEPQESEHDFFIIGHTSTSVSLASGLAKGRDLTGGNENIIAVIGDGS LSGGEAFEGLDYVAELGTNMIIIVNDNQMSIAENHGGLYKNLKELRDSNGQCECNFFKAMGLDYIYVNDGNDVQALIEAFSKVKDIQHPIVVHINTLKGKGYERAEQDKETYHWRTPFNPETGEAKVNYEEEDYSEVTAQYLLKKM EEDSRVVTITSGTPAVLGFTPDRRKEAGKQFVDVGIAEEHAVALASGIAANGGKPVYGVYSTFIQRSYDQLSQDLCINNNPAVLLLVFWGTLSGMNDVTHLCFFDIPLISNIPNMVYLAPTCKEEYLAMLEWSIRQNEYPVAIRVPA TDVITCGEPVETDYSVLNRYKVTHRGSKVAILALGSFYGLGQSVASLLKEKANIDATLINPRYITGVDNELMDELKAADHELVITLEDGVLDGGFGEKIARYYGATNMKVLNFGAKKEFVDRYDIQEFLRANHLTDEQIVEDITAVIG

[0075] The small fragment between the Nde I and BamHI restriction sites of the pET28a plasmid (Novagen) was replaced with the double-stranded DNA molecule corresponding to the sequence shown in ADS1-12 (SEQ ID NO:1-12) to obtain the recombinant plasmid pET28a::ads (which has been verified by sequencing).

[0076] The recombinant plasmid pET28a::ads was introduced into E. coli BL21(DE3) to obtain the recombinant strain BL21(DE3)::pET28a-ads.

[0077] The recombinant bacteria BL21(DE3)::pET28a-ads were cultured in LB liquid medium containing 50 μg / mL kanamycin until the bacterial culture reached OD. 600nm The value is about 0.4. After cooling at 4℃ for about 15 minutes, IPTG with a final concentration of 0.05mM is added, and the protein expression is induced by low temperature at 16℃ and 180rpm for 12-16 hours.

[0078] Bacterial cells expressing ADS protein were harvested, resuspended in phosphate buffer, sonicated, and then incubated with 3-oxoDCA and pyruvate at 37°C for 2 hours. A control group was set up for boiling inactivation. The remaining content of 3-oxoDCA was detected by mass spectrometry. The results are shown in Table 1.

[0079] Table 1. Residual amount of 3-oxoDCA after ADS incubation

[0080]

[0081]

[0082] Example 2: Isolation and Identification of Two-tailed Acid Compounds

[0083] 1. Compound transformation and extraction

[0084] Under the conditions of Example 1, ADS-1 achieved the highest conversion rate of 3-oxoDCA, and ADS-1 was selected for further separation and identification of the catalytic product. Following the scale-up method of Example 1, 100 mg of 3-oxoDCA was converted. The reaction system was extracted with ethyl acetate to obtain an extract, which was then distilled under reduced pressure to obtain a crude extract.

[0085] 2. Compound isolation and purification

[0086] The fermentation product was extracted and concentrated, and the 3-acetone deoxycholic acid compounds were separated by HPLC-ELSD. The separation conditions were as follows: a Cosmosil 5C18-MS-II column (4.6 mm × 250 mm) was used, the column temperature was 35 °C, and the flow rate was 1.0 mL / min. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution. Compound I was purified by isocratic elution under mobile phase A:B = 60:40 (v / v). After concentration and drying under reduced pressure, the product was dissolved in 600 μL of methanol solution and allowed to stand at 4 °C for 5 days, resulting in the precipitation of colorless crystals, yielding 3-acetone deoxycholic acid.

[0087] 3. Compound structure identification

[0088] Compound 3-acetone deoxycholic acid is a white powder with an HRESIMS m / z of 433.2957 [MH]. - , hydrogen spectrum ( 1 H-NMR and carbon spectroscopy (H-NMR) 13 The C-NMR spectra are shown in Table 2. Based on the physicochemical properties, 1H NMR, and 1C NMR data, the compound was identified as 3-acetone deoxycholic acid (compound I). The structural characterization data of compounds II-VIII are shown in Figures xx-xx.

[0089] Table 2 NMR data for 3-acetylketodeoxycholic acid

[0090]

[0091]

[0092] Example 3: Detection of the production of bi-tailed acids by various intestinal symbiotic bacteria

[0093] Materials: Bacteroides (including Bacteroides coli, Bacteroides esculenta, Bacteroides cusiae, Bacteroides cusiae, Bacteroides foetida, Bacteroides fragilis, Bacteroides foetida, Bacteroides koreanum, Bacteroides ovalis, Bacteroides polymorpha, Bacteroides xylana), Clostridium (including Clostridium neonatale, Clostridium putrefactiveum), Enterococcus (including Enterococcus staphylococcus, Enterococcus faecalis, Enterococcus lactis), Escherichia (including Escherichia coli), Eubacterium (including Eubacterium inertia), Trichophyton (including Trichophyton spp.), Lactobacillus (including Lactobacillus baltii, Lactobacillus reuteri), Lactobacillus (including Lactobacillus fermentum, Lactobacillus gasseri), Parabacterium (including Parabacterium difficile), Paraclobroma (including Paraclobroma sosei), Ruminococcus (including active Ruminococcus), modified GAM medium.

[0094] Methods: The above-mentioned intestinal symbiotic stock solution was inoculated at a 5% inoculation ratio into anaerobic modified GAM liquid medium containing substrate (3-oxoDCA) under anaerobic conditions. The culture was incubated at 37°C and 220 rpm in an anaerobic environment (70% nitrogen; 25% carbon dioxide; 5% hydrogen) for 48 hours. After incubation, 50 μL of the bacterial culture was mixed with 50 μL of methanol, centrifuged at 12000 rpm, and the supernatant was filtered through a membrane. The content of 3-ethione deoxycholic acid was determined by mass spectrometry.

[0095] Results: As shown in Table 3, all listed bacterial species can synthesize 3-ethylhexanoate, with Bacteroides ovalis showing the strongest synthesis ability.

[0096] Table 3. Detection of 3-acetylketodeoxycholic acid content synthesized by various intestinal symbiotic bacteria.

[0097]

[0098]

[0099] Example 4: The promoting effect of 3-acetylketodeoxycholic acid on lactic acid bacteria

[0100] Materials: Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus curlis, Lactobacillus johnsonii, SDM medium

[0101] Methods: The above-mentioned Lactobacillus stock solution was inoculated at a 5% inoculation ratio into anaerobic SDM liquid medium containing / without 3-acetylenedeoxycholic acid under anaerobic conditions. The culture was carried out at 37°C and 220 rpm in an anaerobic environment (70% nitrogen; 25% carbon dioxide; 5% hydrogen) for 48 hours. The OD of the strain was then measured. 600 .

[0102] Results: 3-Ethyl ketone deoxycholic acid increased the OD of the listed bacterial species after 48 hours. 600 The value promotes the growth of lactic acid bacteria. The results are shown in Table 4.

[0103] Table 4. Effects of 3-Ethylhexone deoxycholic acid on the growth of lactic acid bacteria

[0104]

[0105] Note: The statistical method used was a two-tailed independent samples t-test.

[0106] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

Claims

1. A 3-oxo deoxycholic acid compound characterized in that: The compounds are shown in formulas I-VIII:

2. A synthetase of the compound of claim 1, characterized by: The amino acid sequences of the synthases are shown in SEQ ID NO:1-12, respectively.

3. A method for the in vitro enzymatic preparation of a compound according to claim 1, characterized by: The preparation method includes the following steps: 1) Obtain the catalytic enzyme ADS by overexpressing any one of the synthases described in claim 2 in Escherichia coli; 2) The ADS protein was incubated with substrates 3-oxocholic acid (3-oxoCA), 3-oxodeoxycholic acid (3-oxoDCA), 3-oxochenodeoxycholic acid (3-oxoCDCA), 3-oxolithocholic acid (3-oxoLCA) and pyruvate at 37°C for 10-16 hours. 3) The 3-acetone deoxycholic acid compounds were separated by HPLC-ELSD.

4. The process for the microbiological fermentation of the compound according to claim 1, characterized by the fact that: The preparation method includes the following steps: 1) Activate one or more of the following strains: Bacteroides, Clostridium, Escherichia, Eubacterium, Trichophyton, Lactobacillus, Lactobacillus, Parabacterium, Paraclostridium, or Ruminococcus, and inoculate them into the fermentation seed culture medium. 2) Transfer the fermentation seed liquid to a fermentation medium containing substrates 3-oxoCA, 3-oxoDCA, 3-oxoCDCA, and 3-oxoLCA, and anaerobic ferment for 48 h; 3) Extract and concentrate the fermentation product, and separate the 3-acetone deoxycholic acid compounds using HPLC-ELSD.

5. The method of claim 4, wherein: The *Bacteroides* genus includes *Bacteroides foetida*, *Bacteroides escherichia*, *Bacteroides foetida*, *Bacteroides nigra*, *Bacteroides foetida*, *Bacteroides fragilis*, *Bacteroides foetida*, *Bacteroides koreanum*, *Bacteroides ovalis*, *Bacteroides pleomorphica*, or *Bacteroides xylana*; the *Clostridium* genus includes *Clostridium neonate* or *Clostridium putrefactive*; the *Enterococcus* genus includes *Enterococcus durableis*, *Enterococcus faecalis*, or *Enterococcus lactis*; the *Escherichia* genus includes *Escherichia coli*; the *Eubacterium* genus includes *Eubacterium indolentum*; the *Trichophyton* genus includes *Trichophyton styloides*; the *Lactobacillus* genus includes *Lactobacillus balolatus* and *Lactobacillus reuteri*; the *Lactobacillus* genus includes *Lactobacillus fermentum* and *Lactobacillus gasseri*; the *Parabacteroides* genus includes *Parabacteroides digiri*; The *Paracobacterium* genus includes *Paracobacter spp.*; or the *Ruminococcus* genus includes *Active Ruminococcus*.

6. Use of the compound of claim 1 in the preparation of products that promote the growth of lactic acid bacteria.

7. Use according to claim 6, characterized in that: The lactic acid bacteria include Lactobacillus genus, Lactobacillus helveticus, Lactobacillus curvatureii, and Lactobacillus johnsonii.

8. Use according to claim 6, characterized in that: The product is used for disease intervention.

9. Use according to claim 8, characterized in that: The disease in question is metabolic-associated fatty liver disease (MAFLD).

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