Enzymatic synthesis method of plant growth promoter thienodolin and its application

Abstract

The present invention provides a method for synthesizing the plant growth promoter thienodolin from 6-Cl-Trp. The method uses ThnJ, ThnF, ThnE, ThnL, ThnM, ThnD, ThnC, ThdO, and ThnA proteins to co-catalyze the synthesis of thienodolin. This method completely reconstructs the biosynthetic pathway of thienodolin for the first time, elucidates the catalytic function of the biocatalytic enzyme, and achieves a one-pot synthesis from 6-Cl-Trp (6-chloro-tryptophan) to thienodolin.

Description

Technical Field

[0001] The invention belongs to the technical field of thienodolin synthesis, and particularly relates to an enzymatic synthesis method of thienodolin, a plant growth promoter, and application thereof. Background Art

[0002] Thienodolin (abbreviated as THN, chemical structure see Figure 1 THN is a natural product containing an indolethiophene skeleton discovered in the 1990s. It is metabolized by Streptomyces albogriseolus MJ286-76F7. THN has the biological activity of promoting the growth of young shoots and roots in germinating seeds in a concentration-dependent manner.

[0003] Researchers at home and abroad have conducted research on the synthesis of THN. While chemical synthesis of THN and its derivatives has been achieved, this approach suffers from cumbersome steps and low yields. Regarding biosynthesis, a complete synthetic pathway and route for THN has yet to be reported, and efficient in vitro production using enzymes is still unavailable. Summary of the Invention

[0004] The present invention aims to provide a complete biosynthetic pathway for the plant growth promoter thienodolin, which utilizes a bioenzymatic method to realize an in vitro synthesis strategy of THN and utilizes a multi-enzyme cascade reaction method to catalyze the bioconversion of 6-Cl-Trp (6-chloro-tryptophan) into thienodolin (THN).

[0005] The present invention provides a method for synthesizing thienodolin from 6-Cl-Trp, which uses ThnJ, ThnF, ThnE, ThnL, ThnM, ThnD, ThnC, ThdO and ThnA proteases to synthesize thienodolin under in vitro catalysis.

[0006] The method of the present invention, wherein the synthesis of 6-Cl-TLA from 6-Cl-Trp is carried out in the presence of ThnJ, ThnF, ThnE, ThnL and ThnM proteases;

[0007] The process from 6-Cl-Trp to 6-Cl-IPA is catalyzed by aminotransferase (ThnJ), which converts the amino group of 6-chloro-tryptophan into a ketone group.

[0008] From 6-Cl-IPA to 6-Cl-TPA, the process is completed by sulfotransferase (ThnF) with the participation of sulfotransferase (ThnL) and sulfotransferase kinase (ThnM);

[0009] Furthermore, from 6-Cl-TPA to THN, ThnE, ThdO, ThnD, ThnC, and ThnA participated in the catalytic process;

[0010] The process from 6-Cl-TPA to 6-Cl-TLA is catalyzed by short-chain dehydrogenase (ThnE).

[0011] The process from 6-Cl-TLA to 6-Cl-THA is completed with the participation of oxidoreductase (ThdO), cytochrome P450 enzyme (ThnC) and ThnD.

[0012] From 6-Cl-THA to thienodolin, the process is catalyzed by the aminotransferase ThnA.

[0013] The amino acid sequence of ThnJ protease is SEQ ID NO: 1, the amino acid sequence of ThnF protease is SEQ ID NO: 2, the amino acid sequence of ThnM protease is SEQ ID NO: 3, the amino acid sequence of ThnL protease is SEQ ID NO: 4, the amino acid sequence of ThnE protease is SEQ ID NO: 5, the amino acid sequence of ThnD protease is SEQ ID NO: 6, the amino acid sequence of ThdO protease is SEQ ID NO: 7, the amino acid sequence of ThnC protease is SEQ ID NO: 8, and the amino acid sequence of ThnA protease is SEQ ID NO: 9.

[0014] As a record of one embodiment of the present invention, the amino acid sequences of ThnJ, ThnF, ThnE, ThnL, ThnM, ThnD, ThnC, ThdO and ThnA proteases are as follows:

[0015] SEQ ID NO: 1:

[0016] MPTNQVPPAPLRTTEISRLPAYRADPPAKHADGLLDLANNELLTLPPLPAVVESLRLSARVHLYPDPTARTLRQAVARHFATDPEEIVVGPGSGAVLHQLLLALCGPGDEVLYPWPGFDAYPFLITTAGAQGTPVPLTPSGEHDLSAFAAKVDTRTRIVVLCSPHNPTGRRIPRTELAAFL GSLPPHVVTVLDQAYVEFDEHEETNHLDLLRTAPRLVLLRTFSKAYGLAGLRAGYALAAPDLAGLAYKTLLPFSVTRTAEHAAQVSLEQHGQLAARLDTVRRGRSLSAGLRAEGLDPLPSFGNFLWLPLGAATERFAQVTLAAGVRVRAYPGAGVRITVGGDEAHRRVLDAARLFRTADT;

[0017] SEQ ID NO:2:

[0018] MPESGTAQARTPRTAHPAQPDKKAPAPVSPFSAITAIVADADVFTEFIEDAVAQYRLGLASVAAGGSRLHRPNSFGLLGGTLDGDALTVRRVAFAANVRAVDDVPLEEFRDTIVPRFGKQYDDGERGFWCDSRQLLKVVREFEAADLEMLGSVHMHPDWHRVGPPHERRELLSENPSRMDEYLFRSAGWPLNIICYLESRGSGVTHTFAAWKPPSADGERAARTPIRFFARGHEDA;

[0019] SEQ ID NO:3:

[0020] VSLPPLVEPADELTVDEVRRYSRHLIIPDVGMDGQKRLKNAKVLCVGAGGLGSPALMYLAAAGVGTLGIVEFDEVDESNLQRQIIHSQADIGRSKAESARDTVKGINPYVNVILHEERLEADNVMDIFSQYDLIVDGTDNFATRYLVNDACVLLNKPYVWGSIYRFDGQASVFWSEHGPCYRCLYPEPPPPGMVPSCAEGGVLGVLCASIGSIQVNEAIKLLAGIGEPLVGRLMIYDALEMQYRTVKVRKDPDCAVCGENPTVTELIDYEAFCGVVSEEAQEAAAAGSTITPKQLKEWIDDGENIEIIDVREQNEYEIVSIPGAKLIPKNEFLMGSALEGLPQDKKIVLHCKTGVRSAEVLAVLKSAGFADAVHVGGGVIGWVNQIEPHKPVY;

[0021] SEQ ID NO:4:

[0022] MSVTVRIPTILRTYTGGQAEVAAEGATLAEVIADLEKNHTGISARVLDDQGKLRRFVNVYVNDDDVRFEQGLETATPEGAGVSIIPAVAGG;

[0023] SEQ ID NO:5:

[0024] MTASTRRVAIVTGGTRGIGAALAQRLADSGVDLVVGYAQDSSSADALVRRIEKATAVRVAVRGDIARAETVDALFATADKEFGGVDIVIGCAGAHARRRGPLAETDDADLRHVVDVNLLGTCRLLRAAARHVRPGGRVLAFSSSMALGVPGQAVYNASKAAVEVLVRHLARELAGRDVTVNAVAPGPTGTDLFLRGRTPEDIEALAQQVPLGRIGRPEDVADLVSFLVGPAGGWINGQVVRSNGGIV;

[0025] SEQ ID NO:6:

[0026] MNTTAAPSLYRYDELARLSRDLPVIERIAAWCEEFLCRHPGPGLGRGGSVCPYMPRALAANHVAFTVVRTQGRTRGAIDEAIAAHRTAFLAMEPTSGPGSLDKAIVVILPDVAEEDAAELVDETHRRLKASFVASGLMIGKFHPRSAQGGLHNPGFRPLRSPVPLLAIRHMVDSDLPFLNRPDDPVSDRIGFLQAYERRFEGAEGSPWAARGQAALGEITEEER;

[0027] SEQ ID NO:7:

[0028] MDRYPRNAGTVVSPADPDYERARRIWNNRFDCRPRAIMYPRDEAEAAAALGYARDNEVPFRIRSGGHNSEGFCTVDDGIIIDLSKLGSIAVNNDRTRAVISPGVMLGEVYANLWSVGGTIPAGVCPDIRIGGHVLGGGIGMLVRRSRGLLIDNLVGLTVVDARGERLQVDADHHPDLFWACKGGGGGNYGIATSYTFEMRPISDVTIFKLQWDWQGGMAVLDAWQHWLLSADSRVNARFNVFSSTVGTVMTVGLFEGSADELKSILQPLVEKFPPRDSFVRTMPYMECVGTFSEQVPSIRAKFVPALSAGPLDADALKTLERWHKDAPTGVKTGLYGLGGEVLGSVSPDSSAFAHRDASICVEYLGHWQGAEHDREHLGWLSGVREEMDAFMTGGAYVNSPDRDLENWLHAYYGESLPRLMDVKRRYDPDNVFAFEQSIPGSLTPEARAAGLTDTVVEDLRARGLSA;

[0029] SEQ ID NO:8:

[0030] MAPEAFPFPDEPLGEVPRSCAWRRAHDPLGDAVLPSGDVVRVAVRFADVEAVLTDPRFSRDLNRPGSPRLQPDADMSEDRDTLINLDPPRHTRLRRILSRPFSVRGSEAWRPRIRAIAQAFVDDMVAAGPPADLMTALARQLPIRVIAEILGVGDCDLDRFRDWSETAMTIGPDRAAARAKGLTEFYAYLTDLVERHRREPGTDLLDAMIAARDGEDRLTEAELIDTARSLLLAGHETTMTTLGRGVFSLLRHRDQYEDLVADPELVVPAVEEILRHDFPADVGFLRVAQEDVDLPSGRVAKGQGVMPLISSAHRDERRCPDPDRFDIHRPSPTHLAFGKGPHYCIGAHLARIQLHEALSILVHDLPGLALAVPADEVPWQPGMVTHALACLPITW;

[0031] SEQ ID NO:9:

[0032] .

[0033] The present invention completely reconstructs the biosynthetic pathway of THN for the first time, clarifies the catalytic function of the biocatalytic enzyme, and realizes the one-step synthesis from 6-Cl-Trp to THN through a "one-pot method". BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 : Schematic diagram of the in vitro enzymatic synthesis pathway of THN.

[0035] Figure 2 :6-Cl-TLA preparative liquid phase detection results and 6-Cl-TLA mass spectrometry results.

[0036] Figure 3 :6-Cl-THA preparative liquid phase detection results and mass spectrometry results of 6-Cl-THA.

[0037] Figure 4 : THN preparation liquid phase detection results and THN mass spectrometry results. DETAILED DESCRIPTION

[0038] The invention realizes the in vitro one-pot enzymatic synthesis of 6-Cl-TLA, 6-Cl-THA and THN starting from 6-Cl-Trp. 6-Cl-Trp is used as a substrate, and ThnJ (aminotransferase, SEQ ID NO: 1), ThnF (sulfur transferase, SEQ ID NO: 2), ThnL (sulfur carrier protein, SEQ ID NO: 3), ThnM (sulfur carrier protein kinase, SEQ ID NO: 4) and ThnE (short-chain dehydrogenase, SEQ ID NO: 5) and their required cofactors pyridoxal phosphate (PLP), sodium pyruvate, magnesium chloride, sodium thiosulfate, adenosine triphosphate (ATP), dithiothreitol (DTT) and reduced coenzyme II (NADPH) are added to achieve the synthesis of 6-Cl-TLA (3-(6-chloro-1H-indol-3-yl)-2-mercaptopropanoic acid).

[0039] Based on the synthesis of 6-Cl-TLA, the addition of ThnD (SEQ ID NO: 6), ThdO (FAD-dependent oxidoreductase, sequence 7) and ThnC (cytochrome P450 enzyme, SEQ ID NO: 8) can achieve the synthesis of 6-Cl-THA (6-chloro-8H-thieno[2,3-b]indole-2-carboxamide).

[0040] THN can be synthesized by adding ThnA (aminotransferase, SEQ ID NO: 9) and cofactor glutamine to the synthesis of 6-Cl-THA.

[0041] The full names of the abbreviations involved in the present invention are shown in Table 1.

[0042] Table 1: Alphabetical abbreviations

[0043] Abbreviation Full name 6-Cl-Trp (R)-2-amino-3-(6-chloro-1H-indol-3-yl)propanoic acid 6-Cl-IPA 3-(6-chloro-1H-indol-3-yl)-2-oxopropanoic acid 6-Cl-TPA 3-(6-chloro-1H-indol-3-yl)-2-thioxopropanoic acid 6-Cl-TLA 3-(6-chloro-1H-indol-3-yl)-2-mercaptopropanoic acid 6-Cl-THA 6-chloro-8H-thieno[2,3-b]indole-2-carboxamide THN Thienodolin

[0044] The amino acid sequences of the ThnJ, ThnF, ThnE, ThnL, ThnM, ThnD, ThnC, ThdO and ThnA proteases used in the present invention are SEQ ID NOs: 1-9, respectively; however, other homologous proteases can also be used, such as ThnJ can be replaced by Ind8 (GenBank: AJT38689.1), Cxm7 (GenBank: AVL27082.1) and MarG (GenBank: AHF22859.1), etc. The homologous proteases have the same or similar effects as the initial proteases.

[0045] Example 1: One-pot synthesis of 6-Cl-TLA

[0046] (1) Inducible expression of proteins

[0047] The engineered E. coli strains expressing ThnJ, ThnE, ThnF, ThnL, and ThnM were activated and cultured on solid LB plates. The single clones grown on the LB solid plates were transferred to LB liquid medium and cultured overnight at 37°C and 220 rpm to prepare seed liquid. The seed liquid was transferred to a 2L conical flask containing 500 mL of TB medium at a 1% inoculum volume and kanamycin was added at a final concentration of 50 μg / mL. The culture was then continued at 37°C and 220 rpm until the OD 600 Between 0.8 and 1, 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added, and then cultured at 18°C and 150 rpm for 18 to 22 h to induce protein expression.

[0048] (2) Protein purification

[0049] The cells were collected by centrifugation and resuspended in Lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 10% glycerol, pH 8.0). The cells were disrupted by ultrasonication and centrifuged at high speed (10,000 × g, 4 ° C). The supernatant was filtered by Ni 2+ -NTA affinity column, rinse with Wash buffer (50mM NaH2PO4, 300mM NaCl, 20mM imidazole, 10% glycerol, pH 8.0) until no protein can be detected in the effluent by Coomassie Brilliant Blue G-250 method, and elute the target protein with Elution buffer (50mM NaH2PO4, 300mM NaCl, 250mM imidazole, 10% glycerol, pH 8.0). According to the size of the protein, an ultrafiltration tube of appropriate size is selected to concentrate the protein solution. Finally, a PD-10 desalting column is used to remove imidazole, and the buffer solution uses Desalting buffer (50mM NaH2PO4, 300mM NaCl, 10% glycerol, pH 8.0).

[0050] (3) One-pot reaction

[0051] The reaction buffer was NaH2PO4 solution (50 mM, pH 8.0), 750 μM 6-Cl-Trp was used as the substrate, and final concentrations of 10 μM ThnJ, 10 μM ThnE, 10 μM ThnF, 10 μM ThnM, 10 μM ThnL, 20 μM pyridoxal phosphate, 5 mM DTT, 10 mM NADPH, 2 mM ATP, 2 mM Na2S2O3, 5 mM MgCl2 and 3 mM sodium pyruvate were added. The reaction was carried out at 30°C for 4 hours, and then ethyl acetate was added to the reaction system twice with twice the volume of the reaction solution for extraction. The extracted organic phase was rotary evaporated and dissolved in an appropriate amount of methanol. The reaction samples were analyzed by high performance liquid chromatography (HPLC), and the test results are shown in FIG. Figure 2 The new product was detected, and the conversion efficiency was about 98%. High-resolution mass spectrometry (HR-LCMS) analysis confirmed that the new product was 6-Cl-TLA ([M+H] + The theoretical value is 256.0199, and the measured value is 256.0187).

[0052] Example 2: One-pot synthesis of 6-Cl-THA.

[0053] (1) Inducible expression of proteins

[0054] Escherichia coli engineered strains expressing ThnJ, ThnF, ThnE, ThnL, ThnM, ThnD, ThdO, and ThnC were activated and cultured on solid LB plates. Single colonies grown on the LB solid plates were transferred to LB liquid medium and cultured overnight at 37°C, 220 rpm to prepare seed solution. The seed solution was transferred to a 2L conical flask containing 500 mL of TB medium at a 1% inoculum volume and kanamycin was added at a final concentration of 50 μg / mL. The culture was continued at 37°C, 220 rpm until the OD 600 Between 0.8 and 1, 0.2 mM IPTG was added, and then cultured at 18°C and 150 rpm for 18 to 22 h to induce protein expression.

[0055] (2) Protein purification

[0056] The cells were collected by centrifugation and resuspended in Lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 10% glycerol, pH 8.0). The cells were disrupted by ultrasonication and centrifuged at high speed (10,000 × g, 4 ° C). The supernatant was filtered by Ni 2+-NTA affinity column, rinse with Wash buffer (50mM NaH2PO4, 300mM NaCl, 20mM imidazole, 10% glycerol, pH 8.0) until no protein can be detected in the effluent by Coomassie Brilliant Blue G-250 method, and elute the target protein with Elution buffer (50mM NaH2PO4, 300mM NaCl, 250mM imidazole, 10% glycerol, pH 8.0). According to the size of the protein, an ultrafiltration tube of appropriate size is selected to concentrate the protein solution. Finally, a PD-10 desalting column is used to remove imidazole, and the buffer solution uses Desalting buffer (50mM NaH2PO4, 300mM NaCl, 10% glycerol, pH 8.0).

[0057] (3) One-pot reaction

[0058] The reaction buffer was NaH2PO4 solution (50 mM, pH 8.0), with 1 mM 6-Cl-Trp as the substrate, and final concentrations of 10 μM ThnJ, 10 μM ThnE, 10 μM ThnF, 10 μM ThnM, 10 μM ThnL, 10 μM ThnD, 10 μM ThnC, 10 μM ThdO, 20 μM pyridoxal phosphate, 5 mM DTT, 10 mM NADPH, 2 mM ATP, 2 mM Na2S2O3, 5 mM MgCl2 and 3 mM sodium pyruvate were added. The reaction was carried out at 30°C for 4 hours, and then ethyl acetate twice the volume of the reaction solution was added to the reaction system for extraction twice. The extracted organic phase was rotary evaporated and dissolved in an appropriate amount of methanol. The reaction samples were analyzed by high performance liquid chromatography (HPLC), and the detection results are shown in FIG. Figure 3 The new product was detected in the reaction group with a conversion efficiency of about 43%. High-resolution mass spectrometry (HR-LCMS) analysis confirmed that the new product was 6-Cl-THA ([M+H] + The theoretical value is 251.9886, and the measured value is 251.9884).

[0059] Example 3: One-pot synthesis of THN

[0060] (1) Inducible expression of proteins

[0061] Escherichia coli engineered strains expressing ThnJ, ThnF, ThnE, ThnL, ThnM, ThnD, ThnC, ThdO, and ThnA were activated and cultured on solid LB plates. Single colonies grown on the LB solid plates were transferred to LB liquid medium and cultured overnight at 37°C, 220 rpm to prepare seed solution. The seed solution was transferred to a 2L conical flask containing 500 mL of TB medium at a 1% inoculum volume and kanamycin was added at a final concentration of 50 μg / mL. The culture was continued at 37°C, 220 rpm until the OD 600 Between 0.8 and 1, 0.2 mM IPTG was added, and then cultured at 18°C and 150 rpm for 18 to 22 h to induce protein expression.

[0062] (2) Protein purification

[0063] The cells were collected by centrifugation and resuspended in Lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 10% glycerol, pH 8.0). The cells were disrupted by ultrasonication and centrifuged at high speed (10,000 × g, 4 ° C). The supernatant was filtered by Ni 2+ -NTA affinity column, rinse with Wash buffer (50mM NaH2PO4, 300mM NaCl, 20mM imidazole, 10% glycerol, pH 8.0) until no protein can be detected in the effluent by Coomassie Brilliant Blue G-250 method, and elute the target protein with Elution buffer (50mM NaH2PO4, 300mM NaCl, 250mM imidazole, 10% glycerol, pH 8.0). According to the size of the protein, an ultrafiltration tube of appropriate size is selected to concentrate the protein solution. Finally, a PD-10 desalting column is used to remove imidazole, and the buffer solution uses Desalting buffer (50mM NaH2PO4, 300mM NaCl, 10% glycerol, pH 8.0).

[0064] (3) One-pot reaction

[0065] The reaction buffer was NaH2PO4 solution (50 mM, pH 8.0), with 1 mM 6-Cl-Trp as the substrate, and final concentrations of 10 μM ThnJ, 10 μM ThnE, 10 μM ThnF, 10 μM ThnM, 10 μM ThnL, 10 μM ThnD, 10 μM ThnC, 10 μM ThdO, 10 μM ThnA, 3 mM glutamine, 20 μM pyridoxal phosphate, 5 mM DTT, 10 mM NADPH, 2 mM ATP, 2 mM Na2S2O3, 5 mM MgCl2 and 3 mM sodium pyruvate were added. The reaction was carried out at 30°C for 4 hours, and then ethyl acetate was added to the reaction system twice with twice the volume of the reaction solution for extraction. The extracted organic phase was rotary evaporated and dissolved in an appropriate amount of methanol. The reaction samples were analyzed by high performance liquid chromatography (HPLC), and the detection results are shown in FIG. Figure 4 The new product was detected in the reaction group with a conversion efficiency of about 31%. High-resolution mass spectrometry (HR-LCMS) analysis confirmed that the new product was THN ([M+H] + The theoretical value is 251.0064, and the measured value is 250.9890).

Claims

1. A method for synthesizing thienodolin from 6-Cl-Trp, characterized in that: The method described is to use ThnJ, ThnF, ThnE, ThnL, ThnM, ThnD, ThnC, ThdO and ThnA enzymes to synthesize thienodolin in vitro; The amino acid sequence of the ThnJ enzyme is SEQ ID NO: 1, the amino acid sequence of the ThnF enzyme is SEQ ID NO: 2, the amino acid sequence of the ThnM enzyme is SEQ ID NO: 3, the amino acid sequence of the ThnL enzyme is SEQ ID NO: 4, the amino acid sequence of the ThnE enzyme is SEQ ID NO: 5, the amino acid sequence of the ThnD enzyme is SEQ ID NO: 6, the amino acid sequence of the ThdO enzyme is SEQ ID NO: 7, the amino acid sequence of the ThnC enzyme is SEQ ID NO: 8, and the amino acid sequence of the ThnA enzyme is SEQ ID NO:

9.

2. The method according to claim 1, wherein The method comprises the steps of synthesizing 6-Cl-TLA from 6-Cl-Trp with the participation of ThnJ, ThnF, ThnE, ThnL and ThnM enzymes.

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