Biosynthesis method and application of a selenium-containing compound

Abstract

The present invention provides a method for synthesizing a selenium-containing compound and its application. The provided method for synthesizing a selenium-containing compound uses a multi-enzyme catalytic system to introduce selenium element into a substrate to form a selenium-containing compound. The multi-enzyme catalytic system includes a sulfur transfer protein, a sulfur carrier protein, and a sulfur carrier protein kinase. The present invention utilizes a multi-enzyme catalytic system derived from microorganisms, including a sulfur transfer protein, a sulfur carrier protein, and a sulfur carrier protein kinase, to achieve the introduction of selenium element into the structure of organic small molecule compounds. The biological enzymes used in the present invention can be directly obtained by microbial fermentation. The process of introducing selenium element has the characteristics of being green and efficient, and is an important solution strategy for the synthesis strategy of selenium-containing compounds. The catalytic system mediated by the sulfur carrier protein is typically representative. According to the substrate selectivity and difference of the sulfur carrier protein system, selenium element can be introduced into different small molecule compounds, thereby different selenium-containing compounds can be obtained.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosynthesis of selenium-containing compounds, and specifically relates to a method for introducing selenium elements into the structure of compounds by using sulfur transfer proteins, sulfur carrier proteins, and sulfur carrier protein kinases. Background Art

[0002] Metal elements endow organic compounds with different geometric structures and kinetic properties. Compared with traditional carbon-based compounds for developing new therapeutic drugs, metal-organic compounds have unique advantages. The molecular design of selenium-containing organic compounds is an important strategy for developing anti-cancer and antibacterial drugs. Currently, many selenium-containing organic compounds with biological activities have been synthesized, which can be used as potential anti-tumor agents, antioxidants, antibacterial agents, antiviral drugs, and cytokine inducers.

[0003] Selenium-containing organic compounds are divided into organically synthesized selenium-containing compounds and selenium-containing natural metabolites. There are many types of organically synthesized selenium-containing compounds, including diselenides, selenols, selenic acids, and selenite esters, etc. Regarding selenium-containing natural metabolites, organisms can introduce selenium elements into amino acids or sugars to form selenic acids and selenoglycans. Selenic acids include selenomethionine, selenocysteine, and methylselenocysteine, etc., which can be modified by organisms to produce different types of selenium-containing natural metabolites, such as dimethylselenone, methylselenol, and dimethyldiselenide, etc. So far, there are few reports on the biocatalytic enzymes for introducing selenium elements into selenium-containing organic compounds and their introduction mechanisms. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for synthesizing and applying selenium-containing compounds, which can introduce selenium elements into the structure of compounds, thereby making up for the deficiencies of the prior art.

[0005] The present invention relates to a multi-enzyme catalytic system, including sulfur transfer proteins (Cxm3 and its isoenzymes), sulfur carrier proteins (Cxm4 and its isoenzymes), and sulfur carrier protein kinases (CxmM and its isoenzymes).

[0006] The sulfur transferase Cxm3 has an amino acid sequence as follows:

[0007] MAGVKDAQYVTAATDDGLGGTADSAALLDDLPVTVRFEIEPVRRFLSSALGEYQKCLDSRDADG

[0008] VPSHLPRASGLLFGQVGGAEIVISDVEFVPNVRDSDESVMAEFEATIAPQFGDVYKNPGRGFWSDEQG

[0009] VLQAIRQQSANGLELLGSIHSHPNWHEIGPPHERRQRLSEHPTQMDEYLFRQSCWPVNVIWYVHESSG

[0010] GIAHRVAAWRPGAEQCDRLDIRIPAAIHEQFEVLLEEE(SEQ ID NO:2);

[0011] The sulfur carrier protein Cxm4 has an amino acid sequence as follows:

[0012] MPDVKLPAAFHVLTGGRRQLPVEGANIREVLVGLDQTCPGVLERLMDQEGSVKRYVNVYRNDSDIRSLDGLETKVEHHDVIWIVPAVAGG(SEQ ID NO:3);

[0013] The sulfur carrier protein kinase CxmM has an amino acid sequence as follows:

[0014] VSLPPLVEPAAELTVDEVRRYSRHLIIPDVGMDGQKRLKNAKVLCVGAGGLGISPALMYLAAAG

[0015] VGTLGIVEFDEVDESNLQRQIIHSQADIGRSKAESAKDSVLGINPYVNVILHEERLEAENVMDIFSQY

[0016] DLIVDGTDNFATRYLVNDACVLLNKPYVWGSIYRFDGQASVFWSEHGPCYRCLYPEPPPPGMVPSCAE

[0017] GGVLGVLCASIGSIQVNEAIKLLAGIGDPLVGRLMIYDALEMQYRQVKVRKDPNCAVCGENPTVTELI

[0018] DYEAFCGVVSEEAQEAALGSTITPKQLKEWIDDGENIDIIDVREQNEYEIVSIPGARLIPKNEFLMGG

[0019] ALQDLPQDKKIVLHCKTGVRSAEVLAVLKSAGFADAVHVGGGVIGWVNQIEPSKPVY(SEQ ID NO:4).

[0020] The multi-enzyme catalytic system provided by the present invention can introduce selenium element (Se) into the substrate to form a selenium-containing compound Se-Trp (3-(1H-indol-3-yl)-2-selenoxopropanoic acid), and the substrates include various different types of substrates recognized by the Cxm system and its corresponding isoenzymes involved in the present invention.

[0021] As a specific description of an embodiment, the substrate is indole-3-pyruvic acid (IPA).

[0022] The method for synthesizing indole-3-pyruvic acid (IPA) is to catalyze tryptophan (Trp) to form indole-3-pyruvic acid (IPA) by aminotransferase (Cxm7).

[0023] The aminotransferase (Cxm7) has an amino acid sequence as follows:

[0024] MNVRFAERSTLRDMRAYRDKESSNAEGSSRFTFDLSSNELVLPPLPTVLAGIEKGLPRLARYPD

[0025] PTARDLTEDIAGHLCVSPDEVAVGPGSAGVLQQILLALCGKGDEVVHGWPGFDAYPLLVAISGATGVH

[0026] VPLTASGGHDLDEIRTRVNARTRVVILCSPHNPTGTVIDQDELHGFLRSLPAHVVAVLDEAYVEFDRG

[0027] ANPPGLPVLLSEHSNTVVLRTFSKAYGLAGLRVGYAAGPRQVMATVRKTAIPFGVTRFAEQAAMLSLR

[0028] SEDELCERLAAVAAAREELTAELRELRLPVLLSRANFVWLPLASAAESFARTAATAGVKVRAFPGHGV

[0029] RISVGEAEAHRTLLAALGRADRGNWF (SEQ ID NO:1).

[0030] The present invention utilizes a multi-enzyme catalytic system derived from microorganisms, including a sulfur transfer protein, a sulfur carrier protein, and a sulfur carrier protein kinase, to introduce selenium elements into the structure of organic small molecule compounds. The bioenzymes used in the present invention can be directly obtained through microbial fermentation. The process of introducing selenium elements is green and efficient, and it is an important solution strategy for the synthesis strategy of selenium-containing compounds. The catalytic system mediated by the sulfur carrier protein is typically representative. According to the substrate selectivity and differences of the sulfur carrier protein system, selenium elements can be introduced into different small molecule compounds, thereby obtaining different selenium-containing compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : Schematic diagram of the ISPA synthesis pathway,

[0032] Figure 2 : LC-MS detection result diagram of ISPA.

[0033] Figure 3 : Detection result diagram of 5-Me-Se-Trp.

[0034] Figure 4 : Detection result diagram of 6-Cl-Se-Trp. DETAILED DESCRIPTION OF THE INVENTION

[0035] The present invention provides a multi-enzyme catalytic system, including a sulfur transfer protein (Cxm3 and its isoenzymes), a sulfur carrier protein (Cxm4 and its isoenzymes), and a sulfur carrier protein kinase (CxmM and its isoenzymes). And the method of catalyzing tryptophan (Trp) to form indole-3-pyruvic acid (IPA) by an aminotransferase (Cxm7); and then synthesizing a selenium-containing compound ISPA (3-(1H-indol-3-yl)-2-selenoxopropanoic acid, Se-Trp) from indole-3-pyruvic acid (IPA) is to use a sulfur transferase (Cxm3), a sulfur carrier protein (Cxm4), and a sulfur carrier protein kinase (CxmM) to transfer selenium atoms to IPA ( Figure 1 ).

[0036] Among them, for the aminotransferase (Cxm7), one amino acid sequence is SEQ ID NO:1; for the sulfur transferase Cxm3, one amino acid sequence is SEQ ID NO:2; for the sulfur carrier protein Cxm4, one amino acid sequence is SEQ ID NO:3; for the sulfur carrier protein kinase CxmM, one amino acid sequence is SEQ ID NO:4. However, Cxm7 (aminotransferase), Cxm3 (sulfur transferase), Cxm4 (sulfur carrier protein), and CxmM (sulfur carrier protein kinase) with other sequences can also be used.

[0037] The present invention will be described in detail below in conjunction with embodiments and the accompanying drawings.

[0038] Example 1: One-pot synthesis of Se-Trp

[0039] 1) Protein induction expression

[0040] The engineered Escherichia coli strains that highly express Cxm7, Cxm3, Cxm4, and CxmM are activated on solid LB plates. The monoclonal colonies grown on the LB solid plates are respectively transferred into LB liquid medium and cultured overnight at 37 °C and 220 rpm to prepare the seed solution. The seed solution is transferred into a 2 L conical flask containing 500 mL of TB medium according to an inoculation amount of 1% and kanamycin with a final concentration of 50 mg / mL is added. It is cultured at 37 °C and 220 rpm until the OD 600 is between 0.8 and 1, 0.5 mM IPTG is added, and then it is cultured at 18 °C and 150 rpm for 18 - 22 h to induce protein expression.

[0041] 2) Protein purification

[0042] The cells are collected by centrifugation respectively and resuspended with Lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 10% glycerol, pH 8.0). After ultrasonic cell disruption, it is centrifuged at high speed (10,000×g, 4 °C). The supernatant passes through a Ni 2+ -NTA affinity column and is rinsed with Wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, 10% glycerol, pH 8.0) until no protein can be detected by the Coomassie Brilliant Blue G-250 method in the effluent. The target proteins are eluted with Elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, 10% glycerol, pH 8.0) respectively. A suitable size ultrafiltration tube is selected according to the size of the protein to concentrate the protein solution. Finally, a PD-10 desalting column is used to remove imidazole, and the buffer solution used is Desalting buffer (50 mM NaH2PO4, 300 mM NaCl, 10% glycerol, pH 8.0).

[0043] 3) One-pot reaction

[0044] The reaction buffer was a NaH2PO4 solution (50 mM, pH 8.0). Using 750 μM Trp as the substrate, 10 μM Cxm7, 10 μM Cxm3, 10 μM CxmM, 10 μM Cxm4, 20 μM pyridoxal phosphate, 2 mM ATP, 2 mM Na2SeO3, 5 mM MgCl2 and 3 mM sodium pyruvate were added. The reaction was carried out at 30 °C for 4 hours. Then, twice the volume of methanol was added, vortexed for 10 min, centrifuged at high speed for 10 min, and the supernatant was aspirated for detection. The detection instrument was a 126-quadrupole time-of-flight high-resolution mass spectrometer (liquid chromatography-mass spectrometry).

[0045] Example 2: One-pot synthesis of Se-Trp

[0046] 1) Induction and expression of proteins

[0047] The engineered Escherichia coli strains highly expressing Cxm3, Cxm4 and CxmM were activated on solid LB plates. The monoclonal colonies grown on the LB solid plates were respectively transferred into LB liquid media and cultured overnight at 37 °C and 220 rpm to prepare seed solutions. The seed solutions were transferred into 2 L conical flasks containing 500 mL of TB medium according to an inoculation amount of 1% and kanamycin with a final concentration of 50 mg / mL was added. The culture was carried out at 37 °C and 220 rpm until the OD 600 was between 0.8 and 1, then 0.5 mM IPTG was added, and subsequently the culture was carried out at 18 °C and 150 rpm for 18 - 22 h to induce protein expression.

[0048] 2) Purification of proteins

[0049] The bacteria were collected by centrifugation respectively and resuspended with Lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 10% glycerol, pH 8.0). After ultrasonic cell disruption, high-speed centrifugation (10,000×g, 4 °C) was carried out. The supernatant was passed through a Ni 2+ -NTA affinity column and rinsed with Wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, 10% glycerol, pH 8.0) until no protein was detected by Coomassie Brilliant Blue G-250 method in the effluent. The target proteins were eluted with Elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, 10% glycerol, pH 8.0) respectively. The protein solutions were concentrated using ultrafiltration tubes of appropriate sizes according to the protein sizes. Finally, a PD-10 desalting column was used to remove imidazole, and the buffer solution used was Desalting buffer (50 mM NaH2PO4, 300 mM NaCl, 10% glycerol, pH 8.0).

[0050] 3) One-pot reaction

[0051] The reaction buffer was a NaH2PO4 solution (50 mM, pH 8.0). Using 750 μM indole-3-pyruvic acid as the substrate, 10 μM Cxm3, 10 μM CxmM, 10 μM Cxm4, 2 mM ATP, 2 mM Na2SeO3 and 5 mM MgCl2 were added at final concentrations. The reaction was carried out at 30 °C for 4 hours. Then, twice the volume of methanol was added, vortexed for 10 min, centrifuged at high speed for 10 min, and the supernatant was aspirated for detection. The detection instrument was an Agilent 1260-Q-TOF high-resolution mass spectrometer (LC-MS). The detection results are shown in Figure 2 , by extracting the ion current of Se-Trp (theoretical molecular ion peak: [M+H] + = 267.9877), the formation of Se-Trp was detected (actual molecular ion peak: [M+H] + = 267.9886).

[0052] Example 3: One-pot synthesis of 5-Me-Se-Trp

[0053] The reaction buffer was a NaH2PO4 solution (50 mM, pH 8.0). Using 750 μM 6-Cl-indole-3-pyruvic acid as the substrate, 10 μM Cxm3, 10 μM CxmM, 10 μM Cxm4, 2 mM ATP, 2 mM Na2SeO3 and 5 mM MgCl2 were added at final concentrations. The reaction was carried out at 30 °C for 4 hours. Then, twice the volume of methanol was added, vortexed for 10 min, centrifuged at high speed for 10 min, and the supernatant was aspirated for detection. The detection instrument was an Agilent 1260-Q-TOF high-resolution mass spectrometer (LC-MS). The detection results are shown in Figure 3 , by extracting the ion current of 5-Me-Se-Trp (theoretical molecular ion peak: [M+H] + = 284.0190), the formation of 5-Me-Se-Trp was detected (actual molecular ion peak: [M+H] + = 284.0192).

[0054] Example 4: One-pot synthesis of 6-Cl-Se-Trp

[0055] The reaction buffer was a NaH2PO4 solution (50 mM, pH 8.0). Using 6-Cl-indole-3-pyruvic acid at 750 μM as the substrate, 10 μM Cxm3, 10 μM CxmM, 10 μM Cxm4, 2 mM ATP, 2 mM Na2SeO3 and 5 mM MgCl2 were added. The reaction was carried out at 30 °C for 4 hours. Then, twice the volume of methanol was added, vortexed for 10 min, centrifuged at high speed for 10 min, and the supernatant was aspirated for detection. The detection instrument was an Agilent 1260 Infinity II Quadrupole Time-of-Flight High-Resolution Mass Spectrometer (LC-MS). The detection results are shown in Figure 4 , and by extracting the ion current of 6-Cl-Se-Trp (theoretical molecular ion peak: [M+H] + = 303.9664), the formation of 6-Cl-Se-Trp was detected (actual molecular ion peak: [M+H] + = 303.9670).

Claims

1. A use of a multi-enzyme catalytic system, characterized in that: The application is to introduce selenium into a substrate to form a selenium-containing compound; the substrate is indole-3-pyruvate; the multi-enzyme catalytic system comprises a sulfur transfer protein, a sulfur carrier protein and a sulfur carrier protein kinase; wherein the amino acid sequence of the sulfur transfer protein is SEQ ID NO: 2, the amino acid sequence of the sulfur carrier protein is SEQ ID NO: 3, and the amino acid sequence of the sulfur carrier protein kinase is SEQ ID NO:

4.

2. A method for introducing selenium into a substrate to form a selenium-containing compound, characterized in that: The method uses a multi-enzyme catalytic system to introduce selenium into a substrate to form a selenium-containing compound; the substrate is indole-3-pyruvic acid; the indole-3-pyruvic acid is converted from tryptophan to indole-3-pyruvic acid by aminotransferase; The amino acid sequence of the aminotransferase is SEQ ID NO: 1; The multi-enzyme catalytic system comprises a sulfur transfer protein, a sulfur carrier protein and a sulfur carrier protein kinase; wherein the amino acid sequence of the sulfur transfer protein is SEQ ID NO: 2, the amino acid sequence of the sulfur carrier protein is SEQ ID NO: 3, and the amino acid sequence of the sulfur carrier protein kinase is SEQ ID NO: 4.

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