A method for biosynthesis of lanatoside c from deacetylcardiacol based on acetyltransferase

Abstract

The application belongs to the field of pharmaceutical chemistry, and particularly relates to a method for biosynthesizing lanatoside C based on acetyltransferase to deacetylcardiopine, the application obtains soluble target protein with a purity of >90%, at least one candidate enzyme can catalyze the generation of lanatoside C, and the product can be confirmed by HPLC and LC-MS. The optimal reaction condition of each enzyme is obtained, and the target conversion rate is >50%. The Km and Vmax values of the best active enzyme are obtained, and the affinity and catalytic efficiency of the enzyme and the substrate are evaluated.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, and specifically relates to a method for biosynthesizing langoside C from deacetylated digoxin using acetyltransferase. Background Technology

[0002] Lanatoside C is a cardiac glycoside extracted from the leaves of the digitalis plant. Its chemical structure consists of digitoxinogen, a trisaccharide chain (containing a terminal glucose molecule with an acetyl group attached), and is a precursor to the well-known emergency drug deslanoside and the oral drug digoxin.

[0003] It is a cardiac glycoside drug whose main functions include enhancing myocardial contractility, improving cardiac pumping function, and treating heart failure. It also slows the heart rate and atrioventricular node conduction, thus controlling the ventricular rate in supraventricular tachycardias such as atrial fibrillation and atrial flutter. It is an important clinical drug.

[0004] Currently, lanugin C is mainly extracted and isolated from plants. However, its extraction is affected by plant resources, and the content of lanugin C in plants is extremely low. Furthermore, it is difficult to separate from structurally similar cardiac glycosides, resulting in high extraction costs and low yields. Moreover, there are significant differences between different batches, making quality control difficult. Researchers later used digoxin or desacetyllanugin as raw materials and employed acetyltransferase catalysis to introduce an acetyl group at a specific position for synthesis. This method avoids the plant resource problem but faces technical challenges such as enzyme catalytic efficiency, selectivity, and scalability.

[0005] To address the aforementioned technical problems, the inventors synthesized, optimized, and screened three enzymes that have a significant effect on promoting the synthesis of lanolin C. Summary of the Invention

[0006] The primary objective of this invention is to provide a method for the biosynthesis of langoside C from deacetylated digoxin using acetyltransferase, the method comprising the following steps: (1) Gene cloning and vector construction: The amino acid sequences shown in SEQ ID No.1~SEQ ID No.3 are reverse translated into DNA sequences, optimized, tagged, sent to the company for synthesis, double enzyme digestion, ligation and transformation, positive clone screening, and the ligation product is transformed into the cloning host; (2) Protein expression and purification: The clone host obtained in step (1) is induced to express, the bacterial cells are collected and broken, the protein is purified by affinity chromatography, and the protein is identified and concentrated; (3) In vitro enzyme activity assay and product identification: Establish reaction system: substrate deacetylated digoxin: 0.1-1.0 mM; acetyl coenzyme A: 1.0-5.0 mM; purified enzyme: 0.1-1.0 mg / mL; buffer: 50 mM Tris-HCl, pH 7.5-8.5, containing 10 mM MgCl2; total reaction volume: 100-500 μL; reaction temperature and time: 1-24 hours at 25-37°C; after the reaction, add an equal volume of methanol or acetonitrile to terminate the reaction, vortex mix and centrifuge to collect the supernatant for analysis.

[0007] Preferably, the analysis in step (3) is performed using HPLC.

[0008] Preferably, the HPLC analysis conditions are as follows: chromatographic column: C18 reversed-phase column (5 μm, 4.6 × 150 mm); mobile phase: phase A is water containing 0.1% formic acid, phase B is acetonitrile, gradient elution is used; flow rate: 1.0 mL / min; detection wavelength: 220 nm.

[0009] Preferably, in the reaction described in step (3), the target peak in the HPLC analysis is confirmed using LC-MS.

[0010] The beneficial effects of this invention are: It yields soluble target proteins with a purity >90%, and at least one candidate enzyme can catalyze the formation of Lanatoside C; the product can be confirmed by HPLC and LC-MS. Optimal reaction conditions for each enzyme are obtained, with a target conversion rate >50%. The Km and Vmax values ​​of the enzymes with optimal activity are obtained, and the affinity between the enzyme and substrate and the catalytic efficiency are evaluated.

[0011] Among the three strains, the enzyme derived from *Nonomura sacchari* showed the best performance, with a catalytic efficiency of approximately 10.2 times that for deacetylated digoxin C, demonstrating strong driving force for the synthetic reaction and excellent substrate specificity. The enzyme derived from *Desmodium styracifolium* also showed good catalytic efficiency for the synthesis of digoxin C, with a catalytic efficiency of approximately 9.3 times that for deacetylated digoxin C, exhibiting good absolute efficiency bias. The enzyme derived from *Polyspora curvatureensis* showed good catalytic efficiency for the synthesis of deacetylated digoxin C (3.12 × 10⁻⁶). - ³) and romaine C (3.27×10 - The catalytic efficiencies of the three enzymes were similar, with the latter being slightly higher, indicating that their catalytic efficiency for the synthesis of langoside C was relatively low. Among the three enzymes, the acetyltransferases derived from *Nonomura sacchari* and *Desmodium styracifolium* exhibited better catalytic performance, demonstrating better catalytic efficiency and synthesis of deacetylated digolin, and showing higher utilization of acetyl-CoA. These are the most promising biocatalysts for achieving efficient and targeted acetylation of deacetylated digolin to langoside C. Detailed Implementation

[0012] The present invention will now be described in detail with reference to specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0013] It should be noted that, unless otherwise specified, the methods described in the following embodiments are all conventional methods, and the reagents described are all commercially available.

[0014] In the following examples, deslanoside is deslanoside (more commonly known clinically as digoxin D), a deacetylated derivative of lanatoside C, a fast-acting cardiac glycoside. It is the main active ingredient in digoxin injection administered intravenously, has a rapid onset of action, and is often used in the resuscitation of acute heart failure.

[0015] In the following examples, Acetyl-CoA refers to acetyl-CoA, a crucial metabolic intermediate molecule, not a drug. It plays a central role in cellular energy metabolism (such as the tricarboxylic acid cycle) and substance synthesis (such as fatty acid synthesis). In the context you provided, it may be a precursor or cofactor involved in the langoside C biosynthesis pathway.

[0016] In the following examples, Lanatoside C is lanatoside C, a natural cardiac glycoside extracted from the foxglove plant. Deslanoside mentioned above is its derivative. Lanatoside C itself can also be used as a drug, but its deacetylated product (i.e., digoxin) is more commonly used clinically.

[0017] The experimental principle of this invention is as follows: The chemical structural difference between langoside C and deacetylated langoside lies in the presence or absence of an acetyl group on the second glycosyl group. This experiment utilizes acetyltransferase, with acetyl-CoA as the acetyl donor, to catalyze the acetylation reaction of the glycosyl group on deacetylated langoside, thereby generating langoside C. Candidate enzymes were obtained through heterologous expression, and a reaction system was constructed in vitro. The product was detected using chromatography-mass spectrometry.

[0018] In the following examples, Km (Michaelis constant) reflects the affinity between the enzyme and the substrate. The smaller the Km value, the stronger the affinity. The unit is mM (millimoles per liter).

[0019] Kcat (conversion number): Reflects the catalytic efficiency of an enzyme. It refers to the maximum number of substrate molecules that each enzyme molecule (or each active site) can convert per unit time. The higher the Kcat value, the faster the "working speed" of a single enzyme molecule. The unit is seconds (s). -¹ (per second).

[0020] Kcat / Km (catalytic efficiency constant): The most important indicator for comprehensively measuring the "perfection" or "overall catalytic efficiency" of an enzyme. It considers both the enzyme's binding affinity to the substrate (1 / Km) and its catalytic activity after binding (Kcat). A higher Kcat / Km value indicates higher catalytic efficiency and better specificity of the enzyme for that substrate. The unit is M. - ¹ s - ¹ or mM - ¹ s - ¹(Note: Units are used interchangeably in the table, but comparing relative sizes does not affect the conclusion).

[0021] Example 1 1. Experimental Materials Substrate: Deslanoside (CAS: 17598-65-1), purity ≥ 95%; Cosolvent: DMSO; Acetyl donor: Acetyl-CoA; Candidate enzyme genes: Synthesized and optimized gene sequences (corresponding to enzymes A, B, and C).

[0022] A: Curvular thermophilic polysporum ( Thermopolyspora flexuosa The amino acid sequence of the source enzyme: MRDVSGRTLADLDPFAIFDAEAARLDAYFATLDEEGWARPSRCRGWSVRDVLAHLAGEELYNHACLDDELEELFARLEREGVTGGYDAFNDWCVRRRRGLPVERVLAEWREKNAETRRRM RELGRDALLPTSVGPYPVGLQTFHYDSEYATHADDVGAPVAEEEREGRLHWRTRVGMFALAERGAAVVRVESADGGVLVRGAGHEVLLSPQMFVEATVGRLPGGAVPAELAAVLRVLA (SEQ ID NO.1) B: Nonomura soybeans ( Nonomuraea glycinis The amino acid sequence of the source enzyme: MGDDLLHELDRFDPFDIFDAEAARLDRFFAGLDADGWRRPSRAAGWSVRDVLGHLAGEELYNQACLDGTLQDLLSLMESEGIRGYNDFNEWCVRQRRDVPVDEVLREWRTKNGDTRRRMR ALGPDALLETMAGPYPVGRQAFHYDSEYATHADDVAAPVAEEEAEGRTRWRAAMGRFALAERESKAQVEQTAEQIWVSAGGVSASLSPPEFVEATVGRLPEGHPLDPRLVSALRCLA (SEQ ID NO.2) C: Dermatophytes fasciatus ( Geodermatophilus dictyosporus The amino acid sequence of the source enzyme: MAVDLPALVADLAAESADLDRVLAALDGAAWATPTPAAGWDVADQVAHLAWFDEAATRAAVDPAGFRAEADALTAHGEGFVDVLAAAHRGRAPADLLAWWRAARREYLRVLGGLDPATSLPWYGPPMSAASSGTA RLMETWAHGQDVVDAVGATREPTDRLRHVAHLGVATRGWSSRVHGEEPPDGPVRVELAVPGGGTWTWGPADAADRVTGPALDFCLLVTQRRHRADLALRATGPAADHWLDVAQAFAGPPGAGRQPSSPAGP (SEQ ID NO.3) Molecular cloning reagents: restriction endonucleases (NdeI, XhoI), T4 DNA ligase, PCR-related reagents, competent cells (DH5α, BL21(DE3)).

[0023] Protein expression and purification reagents: LB medium, antibiotics (penicillin or streptomycin), IPTG, Ni-NTA affinity chromatography resin, protein purification buffers (such as binding buffer, elution buffer).

[0024] Analytical reagents: methanol, acetonitrile (chromatographic grade), formic acid (chromatographic grade), Lanatoside C standard.

[0025] 2. Instruments and Equipment Molecular biology equipment: PCR instrument, electrophoresis apparatus, gel imaging system, constant temperature shaker, centrifuge; Protein purification equipment: ultrasonic homogenizer, high-speed centrifuge, chromatography column, AKTA or similar protein purification system (optional), SDS-PAGE electrophoresis system.

[0026] Enzyme reaction and analysis equipment: constant temperature water bath or temperature-controlled shaker, HPLC system (equipped with C18 reversed phase column and UV detector), LC-MS system.

[0027] General equipment: pH meter, analytical balance, clean bench, -80°C refrigerator.

[0028] 3. Experimental Procedure 3.1. Gene Cloning and Vector Construction (1) Sequence design and synthesis: The provided amino acid sequence was reverse-translated into a DNA sequence using bioinformatics software (such as EMBOSS backtranseq), and optimized according to the codon preference of E. coli. NdeI and XhoI restriction sites were introduced at the 5' and 3' ends of the gene, respectively, and a 6×His tag coding sequence was introduced at the N-terminus or C-terminus.

[0029] (2) Gene synthesis: The optimized gene sequence is entrusted to a professional biotechnology company for whole gene synthesis and cloned into a standard plasmid.

[0030] (3) Double digestion: Use NdeI and XhoI to digest the plasmid containing the target gene and the pET series expression vector (such as pET-28a).

[0031] (4) Ligation and transformation: The target gene fragment purified by enzyme digestion was mixed with the linearized vector, and T4 DNA ligase was added to carry out the ligation reaction. The ligation product was transformed into the cloning host (BL21(DE3) competent cells) and plated on LB plates containing the appropriate antibiotics.

[0032] (5) Screening of positive clones: Select single colonies for colony PCR and plasmid double enzyme digestion verification. Send the preliminarily verified positive clones for sequencing to ensure that the gene sequence is correct.

[0033] 3.2 Protein Expression and Purification (1) Construction of expression strain: The recombinant plasmid with correct sequencing was transformed into the expression host E. coli BL21(DE3) competent cells.

[0034] (2) Induction of expression: Pick a single colony and inoculate it into LB liquid medium (containing antibiotics), and culture at 37°C with shaking until the OD600 is about 0.6-0.8. Add IPTG (final concentration 0.1-1.0 mM) and induce expression at an appropriate temperature (such as 16-25°C) for 16-20 hours.

[0035] (3) Collection and disruption of bacterial cells: Collect bacterial cells by centrifugation and resuspend them in binding buffer. Disrupt the cells by sonication, and collect the supernatant (containing soluble protein) after centrifugation.

[0036] (4) Affinity chromatography purification: Load the supernatant onto a pre-equilibrated Ni-NTA affinity chromatography column. Wash with a buffer containing a low concentration of imidazole to remove contaminating proteins, and then elute the target protein with a buffer containing a high concentration of imidazole.

[0037] (5) Protein identification and concentration: Protein purity and molecular weight were determined by SDS-PAGE. The purified protein was concentrated and buffer replaced using ultrafiltration tubes, and the protein concentration was determined. After aliquoting, the protein was stored at -80°C for later use.

[0038] 3.3 In vitro enzyme activity assay and product identification (1) Establishment of the reaction system: Substrate: Deslanoside: 0.1-1.0 mM (diluted from high-concentration DMSO stock solution, ensuring final DMSO concentration ≤3%); Acetyl-CoA: 1.0-5.0 mM; Purified enzyme: 0.1-1.0 mg / mL; Buffer: 50 mM Tris-HCl (pH 7.5-8.5), containing 10 mM MgCl2; Total reaction volume: 100-500 μL; Reaction temperature and time: 1-24 hours at 25-37°C; (2) Set up controls: set up no enzyme, heat-inactivated enzyme, no substrate, etc. as negative controls.

[0039] (3) Reaction termination: After the reaction is completed, add an equal volume of methanol or acetonitrile to terminate the reaction. After vortex mixing, centrifuge and take the supernatant for analysis.

[0040] (4) HPLC analysis: Chromatographic column: C18 reversed-phase column (5 μm, 4.6 × 150 mm); Mobile phase: Phase A is water (containing 0.1% formic acid), and Phase B is acetonitrile, using gradient elution; Flow rate: 1.0 mL / min; Detection wavelength: 220 nm; Preliminary qualitative analysis was conducted by comparing the retention times of Deslanoside and Lanatoside C standards.

[0041] (5) LC-MS verification: The target peak in the HPLC analysis was confirmed using LC-MS. The molecular weight of Lanatoside C should be 42 Da (the mass of one acetyl group) greater than that of Deslanoside.

[0042] Ion source: ESI positive ion mode Scan range: appropriate m / z range 3.4 Determination of enzyme kinetic parameters For the enzyme with the highest activity, its kinetic parameters (Km and Vmax) were determined. With the Acetyl-CoA concentration fixed and the Deslanoside concentration varied, the initial reaction rate was measured, and the Km and Vmax values ​​were calculated using Lineweaver-Burk plots or nonlinear regression software.

[0043] 4. Results and Data Analysis Protein expression and purification: Obtain soluble target protein with a purity >90%.

[0044] Enzyme activity verification: At least one candidate enzyme can catalyze the production of Lanatoside C, and the product can be confirmed by HPLC and LC-MS.

[0045] Kinetic parameters: Obtain the Km and Vmax values ​​of the enzyme with optimal activity, and evaluate the affinity of the enzyme for the substrate and the catalytic efficiency.

[0046] Table 1. Enzyme activities of *Polyspora curvatureis* on different substrates.

[0047] Table 2. Enzyme activities of *Nonomura sacchariformis* on different substrates in soybean.

[0048] Table 3 Enzyme activities of *Gastrophila reticulata* on different substrates

[0049] Among all three strains, the catalytic efficiency (Kcat / Km) for acetyl-CoA was the highest (0.67, 0.85, and 0.054, respectively). This suggests that the core function or evolutionarily best-adapted substrate of these three enzymes is likely acetyl-CoA, or they are highly associated with acetyltransfer reactions.

[0050] Among the three strains, the enzyme derived from *Nonomura sacchari* showed the best performance, with a catalytic efficiency of approximately 10.2 times that for deacetylated digoxin C, demonstrating strong driving force for the synthetic reaction and excellent substrate specificity. The enzyme derived from *Desmodium styracifolium* also showed good catalytic efficiency for the synthesis of digoxin C, with a catalytic efficiency of approximately 9.3 times that for deacetylated digoxin C, exhibiting good absolute efficiency bias. The enzyme derived from *Polyspora curvatureensis* showed good catalytic efficiency for the synthesis of deacetylated digoxin C (3.12 × 10⁻⁶). - ³) and romaine C (3.27×10 -The catalytic efficiencies of the three enzymes were similar, with the latter being slightly higher, indicating that their catalytic efficiency for the synthesis of langoside C was relatively low. Among the three enzymes, the acetyltransferases derived from *Nonomura sacchari* and *Desmodium styracifolium* exhibited better catalytic performance, demonstrating better catalytic efficiency and synthesis of deacetylated digolin, and showing higher utilization of acetyl-CoA. These are the most promising biocatalysts for achieving efficient and targeted acetylation of deacetylated digolin to langoside C.

[0051] This study provides a reliable candidate enzyme tool and a solid foundation of enzymatic data for the enzymatic synthesis of lanolin C, clarifies the industrial application potential of enzymes derived from *Nonomura sacchari* and *Gastrodinium spp.*, and lays a solid foundation for subsequent enzyme engineering modification and process development.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for biosynthesizing langoside C from deacetylated digolinium using acetyltransferase, characterized in that, The method includes the following steps: (1) Gene cloning and vector construction: The amino acid sequences shown in SEQ ID No.1~SEQ ID No.3 are reverse translated into DNA sequences, optimized, tagged, sent to the company for synthesis, double enzyme digestion, ligation and transformation, positive clone screening, and the ligation product is transformed into the cloning host; (2) Protein expression and purification: The clone host obtained in step (1) is induced to express, the bacterial cells are collected and broken, the protein is purified by affinity chromatography, and the protein is identified and concentrated; (3) In vitro enzyme activity assay and product identification: Establish reaction system: substrate deacetylated digoxin: 0.1-1.0 mM; acetyl coenzyme A: 1.0-5.0 mM; purified enzyme: 0.1-1.0 mg / mL; buffer: 50 mM Tris-HCl, pH 7.5-8.5, containing 10 mM MgCl2; total reaction volume: 100-500 μL; reaction temperature and time: 1-24 hours at 25-37°C; after the reaction, add an equal volume of methanol or acetonitrile to terminate the reaction, vortex mix and centrifuge to collect the supernatant for analysis.

2. The method as described in claim 1, characterized in that, The analysis described in step (3) was performed using HPLC.

3. The method as described in claim 1, characterized in that, The HPLC analysis conditions were as follows: Column: C18 reversed-phase column; Mobile phase: Phase A was water containing 0.1% formic acid, and Phase B was acetonitrile, with gradient elution; Flow rate: 1.0 mL / min; Detection wavelength: 220 nm.

4. The method as described in claim 1, characterized in that, In the reaction described in step (3), the target peak in the HPLC analysis is confirmed using LC-MS.

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