A polypeptide with the function of reversible conversion of functional groups, its coding gene and application

Abstract

The application provides a polypeptide with a reversible functional group conversion function, a coding gene and an application, and belongs to the technical field of bioengineering. The polypeptide is composed of an amino acid sequence shown in SEQ ID NO. 1. The polypeptide provided by the application has a reversible conversion function between a carbonyl functional group and a corresponding hydroxyl functional group in the biosynthesis of alkaloids of the stonecrop family, provides a new technical means for the analysis, regulation and biosynthesis of a target compound of a related metabolic pathway, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a polypeptide with reversible functional group transformation, its encoding gene, and its applications. Background Technology

[0002] Plant secondary metabolites have significant applications in medicine, agriculture, and functional foods, among which alkaloids have attracted widespread attention due to their remarkable pharmacological activities. Amaryllidaceae alkaloids, unique to plants in the Amaryllidaceae family, are a diverse class of natural products with significant activity, showing promising applications in anti-Alzheimer's disease, anti-tumor, and antiviral research. However, the biosynthetic pathways of these alkaloids are complex, involving multiple enzymatic reactions, and the functional analysis of key enzymes remains incomplete, severely hindering the systematic elucidation of their synthetic mechanisms and their application in biomanufacturing.

[0003] Aldo-keto reductases (AKRs) are a class of NADP(H)-dependent oxidoreductases widely distributed in organisms. They typically participate in the reduction reactions of aldehydes, ketones, and related intermediates, playing important roles in plant secondary metabolism, stress responses, and the synthesis of certain natural products. Previous studies have shown that AKR family members participate in the transformation of key intermediates in the synthesis of various alkaloids and aromatic compounds; however, their specific substrate selectivity and catalytic functions vary significantly among different plant species.

[0004] Currently, research on AKR-type enzymes in Amaryllidaceae plants remains limited, especially regarding the lack of systematic identification and functional verification of AKR genes directly related to the synthesis of Amaryllidaceae alkaloids. Existing technologies lack AKR enzyme resources with clearly defined sources and functions that can be used for the transformation of key intermediates in Amaryllidaceae alkaloids, as well as technical solutions for their application in vitro or in heterologous systems. Therefore, there is an urgent need to discover and provide an AKR enzyme derived from Amaryllidaceae plants with a clear catalytic function and its coding sequence, to enrich enzyme resources related to the biosynthesis of Amaryllidaceae alkaloids and to provide new technical means for the synthesis and application of these bioactive natural products. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies. Specifically, existing technologies lack AKR enzyme resources with clearly defined sources and functions suitable for the transformation of key intermediates in Amaryllidaceae alkaloids, and also lack technical solutions for their application in vitro or in heterologous systems. This invention provides a polypeptide with reversible functional group transformation capabilities, its encoding gene, and its applications. This polypeptide catalyzes the reversible transformation between carbonyl and corresponding hydroxyl functional groups during the biosynthesis of Amaryllidaceae alkaloids, providing a new technical means for the analysis and regulation of related metabolic pathways and the biosynthesis of target compounds, and has promising application prospects.

[0006] The first objective of this invention is to provide a polypeptide with reversible functionalization, said polypeptide being selected from any one of (a) to (c): (a) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO.1; (b) A conserved variant polypeptide consisting of one or more amino acids substituted, deleted or added to the amino acid sequence described in (a), and possessing reversible functional activity between the carbonyl and hydroxyl groups of Amaryllidaceae alkaloids. (c) An amino acid sequence having more than 95% identity with the amino acid sequence described in (a), and a polypeptide having reversible functional activity between the carbonyl and hydroxyl groups of Amaryllidaceae alkaloids.

[0007] Preferably, the polypeptide is obtained by artificial synthesis, or by synthesizing its encoding gene and then biologically expressing it.

[0008] A second objective of this invention is to provide a polynucleotide selected from the following (d) to (g): (d) Polynucleotides encoding the polypeptides described in (a) to (c), (e) A polynucleotide encoding a fragment, derivative, or analogue of the polypeptides described in (a) to (c); (f) hybridizes with the polynucleotide sequence defined in (d) or (e) under strict hybridization conditions and encodes a polypeptide with redox function of Amaryllidaceae alkaloids, wherein the polynucleotide sequences have more than 70% identity with each other. (g) has more than 70% identity with the polynucleotide sequence defined in (d) or (e) and encodes a polypeptide that has the same or similar reversible carbonyl-hydroxyl group activity and function as the polypeptides described in (a) to (c).

[0009] Preferably, the polynucleotide further includes variants of the polynucleotide, including substitution variants, deletion variants, or insertion variants.

[0010] Preferably, the polynucleotide is in the form of DNA or RNA, wherein the DNA includes cDNA, genomic DNA or artificially synthesized DNA, and the DNA is in the form of single-stranded or double-stranded strands, and is a coding strand or a non-coding strand.

[0011] A third objective of this invention is to provide a recombinant expression vector containing the aforementioned polynucleotides.

[0012] A fourth objective of this invention is to provide a recombinant host cell characterized by being transformed with the aforementioned recombinant expression vector.

[0013] The fifth objective of this invention is to provide a method for preparing a polypeptide with reversible functional activity between the carbonyl and hydroxyl groups of Amaryllidaceae alkaloids, comprising the following steps: (1) Culture the recombinant host cells described above; (2) Obtaining a polypeptide with reversible interaction activity between the carbonyl and hydroxyl groups of Amaryllidaceae alkaloids from recombinant host cells, wherein the polypeptide is obtained as a crude cell extract or after separation and purification.

[0014] The sixth objective of this invention is to provide the application of the above-mentioned polypeptides, polynucleotides, recombinant expression vectors, or recombinant host cells in the reversible transformation between the carbonyl and hydroxyl groups of Amaryllidaceae alkaloids.

[0015] A seventh objective of this invention is to provide a method for reversible conversion between the carbonyl and hydroxyl groups of Amaryllidaceae alkaloids, the method comprising: In the presence of oxidizing or reducing coenzymes, the aforementioned polypeptide is used to carry out an in vitro enzymatic reaction, causing carbonyl reduction or hydroxyl oxidation of the substrate, thereby generating the corresponding product. Alternatively, in the presence of oxidizing or reducing coenzymes, the recombinant host cells described above are used to induce carbonyl reduction or hydroxyl oxidation of the substrate through in vivo or in vitro reactions, thereby generating the corresponding products. The oxidative or reducing coenzyme is NADP(+) or NADPH; The substrate is N-demethylgalantamine, N-demethylnalidin, galantamine, or nalidin.

[0016] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a polypeptide with reversible functional group transformation, its encoding gene, and its applications. The polypeptide belongs to the aldehyde-ketone reductase (AKR) family, but under different coenzyme conditions, it can catalyze a reversible transformation reaction between carbonyl and hydroxyl functional groups in substrates. This enzyme can catalyze the reversible transformation between carbonyl and hydroxyl groups in specific substrates, exhibiting a clear enzymatic function and good reaction applicability. This invention is the first to obtain and identify an AKR with the above-mentioned catalytic activity from plants of the genus *Amaryllis*, providing a new functional enzyme resource for related secondary metabolic reactions in Amaryllidaceae plants. This enzyme can be stably obtained through recombinant expression, making it suitable for in vitro catalytic reactions and the construction of heterologous biosynthetic systems. It provides a new technical means for the analysis and regulation of related metabolic pathways and the biosynthesis of target compounds, showing promising application prospects. Attached Figure Description

[0017] Figure 1 for Lr AKR purified SDS-PAGE electrophoresis image.

[0018] Figure 2 Recombinant plasmid Lr A schematic diagram of the spectrum of AKR-pET-28a.

[0019] Figure 3 for Lr HPLC detection of the reversible conversion between N-demethylgalanthamine and N-demethylnarwedine catalyzed by AKR. 1 is Lr AKR catalyzes the conversion of N-demethylgalanthamine to N-demethylnarwedine in the presence of oxidized coenzyme. 2 is... Lr AKR catalyzes the formation of N-demethylnarwedine from N-demethylgalanthamine in the presence of a reducing coenzyme. Item 3 is a standard for N-demethylgalanthamine. Item 4 is a standard for N-demethylnarwedine.

[0020] Figure 4 for Lr HPLC detection of reversible conversion between AKR-catalyzed galanthamine and narwedine. 1 is Lr AKR catalyzes the conversion of galantamine to narwedine in the presence of oxidized coenzyme. 2 is... Lr AKR catalyzes the conversion of narwedine to galanthamine in the presence of reducing coenzyme. 3 is a galanthamine standard. 4 is a narwedine standard. Detailed Implementation

[0021] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. However, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Test methods in the following embodiments that do not specify specific conditions are generally operated under conventional conditions. Since they do not involve the inventive point, their steps are not described in detail.

[0022] This invention addresses the current lack of AKR enzyme resources with clearly defined sources and functions for the transformation of key intermediates in Amaryllidaceae alkaloids, as well as the lack of technical solutions for their application in vitro or in heterologous systems. The purpose of this invention is to provide a polypeptide with reversible functional group transformation, its encoding gene, and its applications.

[0023] The polypeptide provided by this invention belongs to the aldehyde-ketone reductase (AKR) family, but it can catalyze a reversible transformation reaction between carbonyl and hydroxyl functional groups of the substrate under different coenzyme conditions.

[0024] To achieve the above objectives, a first aspect of the present invention provides a polypeptide with reversible functionalization, said polypeptide being selected from any one of (a) to (c): (a) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO.1; (b) A conserved variant polypeptide consisting of one or more amino acids substituted, deleted or added to the amino acid sequence described in (a), and possessing reversible functional activity between the carbonyl and hydroxyl groups of Amaryllidaceae alkaloids. (c) An amino acid sequence having more than 95% identity with the amino acid sequence described in (a), and a polypeptide having reversible functional activity between the carbonyl and hydroxyl groups of Amaryllidaceae alkaloids.

[0025] In this invention, a polypeptide possessing the function of reversible transformation between the carbonyl and hydroxyl groups of Amaryllidaceae alkaloids is named... Lr AKR polypeptides include polypeptides consisting of the amino acid sequence shown in SEQ ID NO.1, as well as conserved variant polypeptides based on the above amino acid sequence, their active fragments, derivatives or analogs.

[0026] The term "conservative variant polypeptides, active fragments, active derivatives, and analogs" refers to polypeptides whose amino acid sequences have been altered relative to those shown in SEQ ID NO.1, but which are substantially similar to those shown in SEQ ID NO.1. Lr AKR peptides have the same or similar biological functions, namely, peptides that still possess the activity of catalyzing reversible transformation reactions between carbonyl and hydroxyl functional groups in substrates related to Amaryllidaceae alkaloids.

[0027] The conserved variant polypeptide, active fragment, active derivative, or analogue may be any one or more of the following forms: (1) Based on the amino acid sequence shown in SEQ ID NO.1, one or more amino acid residues are replaced by conserved or non-conserved amino acid residues, preferably by conserved amino acid residues; (2) In the polypeptide shown in SEQ ID NO.1, one or more amino acid residues of chemical groups are replaced by other groups without affecting its catalytic reversible carbonyl / hydroxyl group conversion activity; (3) A fusion polypeptide formed by fusing the polypeptide shown in SEQ ID NO.1 with other compounds or polypeptide sequences, preferably a compound or polypeptide used to improve protein stability, solubility or in vivo half-life, such as polyethylene glycol (PEG) or a tagged polypeptide; (4) A polypeptide formed by fusing an additional amino acid sequence to the N-terminus or C-terminus of the polypeptide shown in SEQ ID NO.1, wherein the additional amino acid sequence includes, but is not limited to, a signal peptide, a secretory peptide, an affinity purification tag, or a reporter protein sequence.

[0028] The polypeptide is obtained through artificial synthesis, or by synthesizing its encoding gene and then expressing it biologically.

[0029] The present invention Lr AKR peptides can be natural peptides, recombinant peptides, or artificially synthesized peptides, preferably recombinant peptides. These peptides can be obtained through natural purification, chemical synthesis, or expression in prokaryotic or eukaryotic host cells using recombinant DNA technology. The host cells include, but are not limited to, Escherichia coli, yeast, insect cells, or plant cells.

[0030] A second aspect of the present invention provides a polynucleotide selected from the following (d) to (g): (d) Polynucleotides encoding the polypeptides described in (a) to (c), (e) A polynucleotide encoding a fragment, derivative, or analogue of the polypeptides described in (a) to (c); (f) hybridizes with the polynucleotide sequence defined in (d) or (e) under strict hybridization conditions and encodes a polypeptide with redox function of Amaryllidaceae alkaloids, wherein the polynucleotide sequences have more than 70% identity with each other. (g) has more than 70% identity with the polynucleotide sequence defined in (d) or (e) and encodes a polypeptide that has the same or similar reversible carbonyl-hydroxyl group activity and function as the polypeptides described in (a) to (c).

[0031] For example, a polynucleotide, said polynucleotide is selected from any of the following: (d) Encoding Lr The polynucleotides of the AKR polypeptide are preferably polynucleotides that encode the amino acid sequence shown in SEQ ID NO.1 to form the polypeptide. (e) Encoding Lr Conserved variants of AKR peptides, active fragments, derivatives, or analogs of polynucleotides; (f) is hybridizable to the polynucleotide sequence defined in (d) or (e) under strict hybridization conditions and encodes a polypeptide having redox function of Amaryllidaceae alkaloids, wherein the polynucleotide sequences have at least 70% identity, preferably at least 80%, more preferably at least 90%; (g) has at least 70% identity with the polynucleotide sequence defined in (d) or (e), and the polypeptide it encodes has similarity to... Lr AKR-like or similar carbonyl / hydroxyl reversible functional polynucleotides with activity and function.

[0032] The polynucleotides described in this invention also include variants of the aforementioned polynucleotides, including substitution variants, deletion variants, and insertion variants, as long as the encoded polypeptide still has reversible carbonyl / hydroxyl group functional activity.

[0033] The polynucleotide can be in the form of DNA or RNA, wherein the DNA includes, but is not limited to, cDNA, genomic DNA or artificially synthesized DNA, and the DNA can be in the form of single-stranded or double-stranded, and can be a coding strand or a non-coding strand.

[0034] The polynucleotide sequence encoding the polypeptide shown in SEQ ID NO.1 may be identical to the corresponding coding sequence or be a degenerate variant. As described herein, a "degenerate variant" refers to a different nucleotide sequence resulting from the degeneracy of the genetic codon, but whose encoded amino acid sequence is identical to that of the polypeptide shown in SEQ ID NO.1.

[0035] A third aspect of the present invention provides a recombinant expression vector containing the aforementioned polynucleotides.

[0036] Exemplary, a recombinant expression vector comprising encoding Lr AKR polypeptide polynucleotides. The aforementioned recombinant vectors refer to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, animal cell viruses, retroviruses, or other vectors well-known in the art. Vectors applicable in this invention include, but are not limited to: expression vectors based on the T7 promoter in bacteria, such as pET-28a; vectors in yeast, such as the YEp series vectors; and MSXND expression vectors in mammalian cells. In short, any vector that can stably replicate and exist within the host cell can be used to construct a recombinant expression vector.

[0037] A fourth aspect of the present invention provides a recombinant host cell, characterized in that it has been transformed into a recombinant expression vector.

[0038] For example, one for expressing LrThe recombinant host cell for the AKR peptide, wherein the host cell of the recombinant expression system can be a prokaryotic or eukaryotic microorganism, including but not limited to *Escherichia coli*, yeast, or other microbial hosts suitable for heterologous expression. The recombinant expression system includes: encoding... Lr The AKR polypeptide consists of a polynucleotide, an expression vector functionally linked to the polynucleotide, and a recombinant host cell containing the expression vector. In a preferred embodiment, the recombinant host cell expresses the obtained... Lr AKR peptides can be purified by means of affinity tags, including but not limited to His tags.

[0039] The fifth aspect of this invention provides a method for preparing a polypeptide with reversible functional activity between the carbonyl and hydroxyl groups of Amaryllidaceae alkaloids, characterized by comprising the following steps: (1) Culture recombinant host cells; (2) Obtaining a polypeptide with reversible interaction activity between the carbonyl and hydroxyl groups of Amaryllidaceae alkaloids from recombinant host cells, wherein the polypeptide is obtained as a crude cell extract or after separation and purification.

[0040] An exemplary method for preparing a polypeptide having reversible carbonyl / hydroxyl group activity of Amaryllidaceae alkaloids, the method comprising the following steps: (1) The culture was transferred to a medium containing the coding. Lr Recombinant host cells of recombinant expression vectors for AKR polypeptides with polynucleotides; (2) Obtaining a polypeptide with reversible interaction activity between the carbonyl and hydroxyl groups of Amaryllidaceae alkaloids from the recombinant host cell, wherein the polypeptide can be obtained as a crude cell extract or after separation and purification.

[0041] This invention utilizes gene recombination technology to enable recombinant host cells to carry codes containing... Lr An expression unit for the AKR polypeptide, enabling the host cell to spontaneously or inducedly express the polypeptide under suitable conditions.

[0042] An "expression unit" refers to a nucleic acid sequence capable of initiating transcription and / or translation in a host cell to produce a specific polypeptide. It typically includes a promoter, a coding sequence, and necessary regulatory elements. The recombinant host cell expresses... Lr The AKR peptide can be obtained by cell disruption to obtain a crude cell extract containing the peptide, or by using conventional protein separation and purification methods in the art to obtain a peptide with high purity. The preparation method of the peptide can be adjusted according to the selected expression vector and host cell type. In addition, it should be understood that the technical principle of synthesizing the peptide of the present invention using a cell-free protein expression system (such as a cell-free system) is similar to that of intracellular expression.

[0043] The sixth aspect of this invention provides the application of polypeptides, polynucleotides, recombinant expression vectors, or recombinant host cells in the reversible transformation between carbonyl and hydroxyl groups of Amaryllidaceae alkaloids.

[0044] A seventh aspect of the present invention provides a method for reversible conversion between the carbonyl and hydroxyl groups of Amaryllidaceae alkaloids, the method comprising: In the presence of oxidizing or reducing coenzymes, polypeptides are used to carry out in vitro enzymatic reactions, causing carbonyl reduction or hydroxyl oxidation of the substrate, thereby generating the corresponding products. Alternatively, in the presence of oxidative or reducing coenzymes, recombinant host cells can be used to induce carbonyl reduction or hydroxyl oxidation of the substrate through in vivo or in vitro reactions, thereby generating the corresponding products. The oxidative or reducing coenzyme is NADP(+) or NADPH; The substrate is N-demethylgalantamine, N-demethylnalidin, galantamine, or nalidin.

[0045] Exemplary, one method of utilizing Lr A method for reversibly carbonyl / hydroxyl group conversion of Amaryllidaceae alkaloids using AKR peptides, performed intracellularly or extracellularly, using N-demethylgalanthamine, N-demethylnarwedine, galanthamine, narwedine, or their structural analogs as substrates, via... Lr A method for catalytically generating the corresponding product from AKR peptides, the method comprising one of the following steps: (a) Provides reversible carbonyl / hydroxyl group conversion functionality Lr AKR peptides undergo in vitro enzymatic reactions in the presence of oxidative or reducing coenzymes, causing carbonyl reduction or hydroxyl oxidation of the substrate to generate the corresponding products. (b) Provided a transfer containing encoding Lr Recombinant host cells of AKR polypeptide polynucleotides, in the presence of oxidative or reducing coenzymes, cause carbonyl reduction or hydroxyl oxidation of the substrate through in vivo or in vitro reactions, thereby generating the corresponding products.

[0046] The method described in this invention can utilize enzymes expressed in vivo by recombinant host cells, or synthesize oxidation / reduction products using extracellular or cell-free systems. Its technical principle is similar to that of in vivo expression.

[0047] The present invention provides a polypeptide and its encoding gene, which can catalyze the reversible conversion between carbonyl and hydroxyl groups in a specific substrate, and has a well-defined function and good reactivity.

[0048] This invention is the first to obtain and identify polypeptides with the aforementioned catalytic activities from plants of the genus *Amaryllis*, providing a new functional enzyme resource for related secondary metabolic reactions in Amaryllidaceae plants. The polypeptides can be stably obtained through recombinant expression, making them suitable for in vitro catalytic reactions and the construction of heterologous biosynthetic systems. This provides a new technical means for the analysis and regulation of related metabolic pathways and the biosynthesis of target compounds, and has promising application prospects.

[0049] The invention is further illustrated in the following examples, but these do not limit the scope of the invention. Some molecular cloning methods and operational details may be adjusted according to the instructions of the reagent, enzyme, or kit provider used, and will not be repeated in the examples.

[0050] Example 1 Lr Obtaining AKR-encoded nucleotides According to the nucleotide sequence information disclosed in this invention, the following methods can be used to obtain... Lr The nucleotide encoding AKR.

[0051] Method 1: Through Lycoris radiata ( Lycoris radiata Obtained by amplification in tissues.

[0052] Fresh Lycoris radiata leaves were used as experimental material, quickly frozen in liquid nitrogen, and thoroughly ground. Total RNA was extracted using an RNA extraction kit (Tiangen), following the kit's instructions. After the extracted RNA passed quality testing, first-strand cDNA was synthesized using a reverse transcription kit (Tiangen) to serve as a template for subsequent amplification reactions.

[0053] Using the synthesized cDNA as a template, specific primers were designed, and the cDNA was amplified by PCR. Lr AKR.

[0054] Primer design: Lr AKR, F-terminal: CCGCGCGGCAGCCATATGTCGAACATCCCTGA (SEQ ID NO.3), Lr AKR, R-terminal: CAGTGGTGGTGGTGGTGGTGTCATTCATCTTCAGTTTCCTC (SEQ ID NO.4); The PCR system is shown in Table 1 below; Table 1 shows the parameters of the PCR reaction system.

[0055] The PCR reaction procedure is shown in Table 2 below; Table 2 shows the PCR reaction procedure parameters.

[0056] Method 2: Based on SEQ ID NO.1 ( Lr The nucleotide sequence information of AKR is used to obtain the target nucleic acid molecule through artificial synthesis. In order to adapt to the target host cell (such as E. coli, yeast or other suitable microorganisms for expression), the nucleotide sequence can be codon optimized before artificial synthesis.

[0057] Example 2 Construction of Recombinant Vector Will Lr The AKR CDS sequence (SEQ ID NO.2) was cloned into a suitable expression vector to construct a recombinant expression vector.

[0058] Will Lr AKR-encoded nucleotides are amplified using templates synthesized artificially or obtained from plant tissues, and then cloned into vectors suitable for expression in *E. coli* or other suitable hosts (e.g., pET-28a) to obtain recombinant expression vectors. Details are as follows: The vector pET-28a(+) was digested using the restriction endonucleases NdeI and XhoI.

[0059] Inactivate the endonuclease according to the instructions for use.

[0060] The digested vector and PCR fragment were mixed, and the vector fragment and gene fragment were ligated using T4 DNA ligase.

[0061] The ligation product was transformed into E. coli competent cells DH5α and screened on LB+ kanamycin solid plates (incubated overnight at 37°C).

[0062] Transformants were inoculated into 5 mL of liquid LB + kanamycin medium and cultured with shaking (37°C, 220 rpm for 8-10 hours). Plasmids were extracted using a plasmid extraction kit.

[0063] The extracted plasmids were subjected to DNA sequencing to screen for those carrying the correct DNA. Lr The plasmid containing the AKR sequence was sequenced using universal primers T7 and T7-ter.

[0064] See Figure 1 As shown, recombinant plasmid Lr A schematic diagram of the spectrum of AKR-pET-28a.

[0065] Example 3: Expression of Recombinant Escherichia coli Lr AKR and its separation and purification The plasmids selected in Example 2 were introduced into suitable host cells, such as Escherichia coli BL21(DE3), and positive clones were screened using kanamycin-resistant plates (Kan+, 100 mg / mL) and cultured overnight at 37°C. Positive clones were obtained through selective culture. Single colonies were picked and added to 5 mL of LB liquid medium (Kan+, 100 mg / mL) and cultured at 37°C and 220 rpm until the OD600 was 0.8. The bacterial culture in 5 mL of LB medium was transferred to 800 mL of 2YT medium (Kan+, 100 mg / mL), and cultured at 37℃ and 220 rpm until the OD600 reached 0.6-0.8. Then, the temperature was lowered to 16℃, and 0.5 mMIPTG was added to induce expression for 16 h. Collect the above-mentioned bacterial culture into a collection bottle, centrifuge at 5500 rpm for 10 min, and collect the bacterial cells; The bacterial cells were fully resuspended in buffer A (50 mM Tris-HCl, 200 mM NaCl, pH 8.0); The resuspended bacterial cells were disrupted using a high-pressure cell disruptor. The resulting lysate was centrifuged at 4 °C and 10,000 rpm for 40 min, and the supernatant was collected. The supernatant was loaded onto a Ni Sepharose 6 Fast Flow nickel column equilibrated with buffer A to adsorb the target protein. Elution was performed stepwise using buffers containing 50 mM, 100 mM, and 300 mM imidazole to remove contaminating proteins and obtain the target protein. The eluted protein solution was concentrated to 500 µL using a Millipore ultrafiltration tube, and the protein concentration was determined using a BCA protein assay kit (Pierce, USA), with bovine serum albumin (2 mg / mL) as the standard. The finally purified protein was flash-frozen in liquid nitrogen and then placed in... Store at 80 ℃ for later use, and analyze protein purity and subunit molecular weight by SDS-PAGE. Results are as follows: Figure 2 As shown, Lr AKR purified SDS-PAGE electrophoresis image.

[0066] The method described in this embodiment can be performed in prokaryotic or eukaryotic hosts, and is not limited to *E. coli*; the expression and purification conditions of the peptide can be optimized according to the host characteristics. The obtained peptide can be used for downstream N-methylation reactions or other applications.

[0067] Example 4 utilizes Lr AKR in vitro catalytic reversible carbonyl / hydroxyl group conversion of Amaryllidaceae alkaloid intermediates Lr AKR peptides can catalyze the reversible carbonyl / hydroxyl group conversion of intermediates in the biosynthesis of Amaryllidaceae alkaloids. In the presence of oxidative or reductive coenzymes, they catalyze the reversible carbonyl / hydroxyl group conversion of intermediates in Amaryllidaceae alkaloids. In this example, N-demethylgalanthamine, N-demethylnarwedine, galanthamine, and narwedine were selected as substrates, and the results were verified under in vitro conditions. Lr Catalytic activity of AKR.

[0068] 1) Standard in vitro enzymatic reaction system (example).

[0069] The total reaction volume is 200 μL, and it includes the following components: The buffer solution was Tris-HCl, pH=7.5; Amaryllidaceae alkaloid substrates, such as N-demethylgalantamine or N-demethylnalvidin; Oxidative or reducing coenzymes, NADP(+) or NADPH; Lr AKR protein (preparation method as in Example 3).

[0070] 2) Reaction conditions and termination The reaction system was incubated at 30°C for 2 hours to allow the carbonyl / hydroxyl group to undergo a reversible reaction. Then, 200 μL of methanol was added to terminate the reaction. Finally, the reaction mixture was centrifuged and filtered to remove protein components, and the reaction supernatant was obtained for subsequent analysis.

[0071] 3) Product Analysis The reaction products could be detected and identified using high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), or other analytical methods. Product analysis was performed using an HPLC-MS system (Agilent 1200, Agilent Technologies, USA), and chromatographic separation was performed using a Waters C18 column (4.6 × 250 mm, 5 μm). The mobile phase consisted of 0.1% formic acid aqueous solution (A) and acetonitrile (B), with a gradient elution program as follows: 0–9 min, fraction B 2%; 9–19 min, fraction B increased to 25%; 19–23 min, fraction B increased to 95%; 23–25 min, fraction B maintained at 95%; 25–29 min, fraction B decreased to 2%. The flow rate was set to 1 mL / min, and the injection volume was 50 μL. Mass spectrometry was performed using an ESI ion source in negative ion mode, with a scan range of m / z 50–500.

[0072] The results showed that, Lr Under the catalytic action of AKR: A reversible interconversion reaction occurs between N-demethylgalanthamine and N-demethylnarwedine. Its reaction route is as follows:

[0073] See Figure 3 As shown, Lr HPLC detection of the reversible conversion between N-demethylgalanthamine and N-demethylnarwedine catalyzed by AKR. 1 is Lr AKR catalyzes the conversion of N-demethylgalanthamine to N-demethylnarwedine in the presence of oxidized coenzyme. 2 is... Lr AKR catalyzes the formation of N-demethylnarwedine from N-demethylgalanthamine in the presence of a reducing coenzyme. Item 3 is a standard for N-demethylgalanthamine. Item 4 is a standard for N-demethylnarwedine.

[0074] A reversible interconversion reaction occurs between galanthamine and narwedine, and the reaction pathway is as follows:

[0075] See Figure 4 As shown, Lr HPLC detection of reversible conversion between AKR-catalyzed galanthamine and narwedine. 1 is Lr AKR catalyzes the conversion of galantamine to narwedine in the presence of oxidized coenzyme. 2 is... Lr AKR catalyzes the conversion of narwedine to galanthamine in the presence of reducing coenzyme. 3 is a galanthamine standard. 4 is a narwedine standard.

[0076] The above results indicate that Lr AKR has the ability to catalyze key intermediate steps in the synthesis of Amaryllidaceae alkaloids.

[0077] SEQ ID NO.1 Lr AKR amino acid sequence: > Lr AKR MSNIPELILTKDSRAMPVVGMGTAAYPFAPENTESAIINAIEIGYRHFDTASLYGSEEPLGKAIVEAQKLGFIKSREELFITSKLWCNEAYPDLVIPAIKKSLRNLKMEYLDLYLIHMPFSTKPDSSPPIPVNGEDIVAMDMEGVWKAMEECQRLGMAKA IGVSNFTVKKLEELLSVANIPPQVNQVEMNPTWQQKKLREYCNVKGIHVTAYSPLGGQDTLISPNMVVKSEVLKEIANARGKTLAQVSLRWVYEQGVSIVVKSFNKERIKKNIEIFDWNLSEDECHRISQIPQCKRVTVETLVSNWKPRPDEFFSEETEDE SEQ ID NO.2 Lr AKR cds sequence: > Lr AKR ATGTCGAACATCCCTGAGTTGATCCTAACCAAAGATTCCCGTGCCATGCCCGTGGTTGGCATGGGCACGGCCGCATATCCATTTGCTCCAGAGAATACAGAGTCAGCCATCATCAATGCAATAGAGATCGGTTATCGTCACTTTGACACTGCTTCGTTGTACGGTTCTGAAGAGCCCCTTGGCAAAGCGATCGTAGAGGCGCAAAAGTTGGGGTTTATTAAGTCACGAGAAGAGCTCTTTATTACTTCCAAGTTGTGGTGCAACGAAGCTTACCCTGATCTCGTCATCCCTGCTATCAAAAAGAGTCTCCGAAATCTAAAAATGGAGTACCTGGATCTCTATCTCATCCACATGCCGTTCAGCACGAAACCGGATTCATCGCCTCCAATTCCGGTGAACGGAGAAGACATTGTGGCCATGGACATGGAGGGAGTATGGAAAGCCATGGAGGAATGCCAAAGGCTTGGGATGGCAAAGGCCATTGGAGTTAGTAATTTCACAGTCAAAAAATTAGAGGAACTGCTCTCTGTAGCCAATATACCCCCACAAGTTAACCAGGTGGAAATGAATCCAACATGGCAGCAGAAGAAGTTGAGGGAGTATTGCAATGTGAAAGGCATTCATGTGACTGCTTATTCTCCTTTAGGAGGTCAAGATACGCTAATTTCACCAAATATGGTTGTGAAATCCGAGGTTTTGAAGGAGATAGCCAATGCAAGAGGAAAGACTCTAGCTCAGGTGTCTCTAAGATGGGTATACGAGCAAGGAGTGAGCATAGTCGTAAAAAGCTTCAACAAGGAGAGAATCAAAAAGAATATTGAAATTTTCGACTGGAACTTAAGCGAAGACGAGTGCCATAGGATAAGCCAAATTCCTCAATGCAAAAGAGTTACAGTTGAGACGCTAGTTTCCAATTGGAAGCCCCGCCCAGATGAATTTTTTTCCGAGGAAACTGAAGATGAATGA While preferred embodiments of the present invention have been described above, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0078] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A polypeptide with reversible functional group transformation, characterized in that, The polypeptide is selected from any one of (a) to (c) below: (a) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO.1; (b) A conserved variant polypeptide consisting of one or more amino acids substituted, deleted or added to the amino acid sequence described in (a), and possessing reversible functional activity between the carbonyl and hydroxyl groups of Amaryllidaceae alkaloids. (c) An amino acid sequence having more than 95% identity with the amino acid sequence described in (a), and a polypeptide having reversible functional activity between the carbonyl and hydroxyl groups of Amaryllidaceae alkaloids.

2. The polypeptide with reversible functional group transformation according to claim 1, characterized in that, The polypeptide is obtained through artificial synthesis, or by synthesizing its encoding gene and then expressing it biologically.

3. A polynucleotide, characterized in that, Selected from the following (d) to (g): (d) A polynucleotide encoding the polypeptides described in claims (a) to (c) of claim 1, (e) A polynucleotide encoding a fragment, derivative, or analog of the polypeptide described in claims (a) to (c) of claim 1; (f) hybridizes with the polynucleotide sequence defined in (d) or (e) under strict hybridization conditions and encodes a polypeptide with redox function of Amaryllidaceae alkaloids, wherein the polynucleotide sequences have more than 70% identity with each other. (g) has more than 70% identity with the polynucleotide sequence defined in (d) or (e), and the polypeptide it encodes has the same or similar reversible carbonyl-hydroxyl activity and function as the polypeptides described in (a) to (c) of claim 1.

4. The polynucleotide according to claim 3, characterized in that, The polynucleotide also includes polynucleotide variants, including substitution variants, deletion variants, or insertion variants.

5. The polynucleotide according to claim 3, characterized in that, The polynucleotide is in the form of DNA or RNA, wherein the DNA includes cDNA, genomic DNA or artificially synthesized DNA, and the DNA is in the form of single-stranded or double-stranded strands, and is a coding strand or a non-coding strand.

6. A recombinant expression vector, characterized in that, It contains the polynucleotide as described in any one of claims 2 to 5.

7. A recombinant host cell, characterized in that, The recombinant expression vector of claim 5 was transferred.

8. A method for preparing a polypeptide with reversible functional activity between the carbonyl and hydroxyl groups of Amaryllidaceae alkaloids, characterized in that, Includes the following steps: (1) Culturing the recombinant host cells as described in claim 7; (2) Obtaining a polypeptide with reversible interaction activity between the carbonyl and hydroxyl groups of Amaryllidaceae alkaloids from recombinant host cells, wherein the polypeptide is obtained as a crude cell extract or after separation and purification.

9. The application of the polypeptide of claim 1, the polynucleotide of any one of claims 2 to 5, the recombinant expression vector of claim 6, or the recombinant host cell of claim 7 in the reversible transformation between the carbonyl and hydroxyl groups of Amaryllidaceae alkaloids.

10. A method for the reversible conversion between the carbonyl and hydroxyl groups of an Amaryllidaceae alkaloid compound, characterized in that, The method includes: In the presence of oxidizing or reducing coenzymes, the polypeptide of claim 1 is used to carry out an in vitro enzymatic reaction to reduce the carbonyl group or oxidize the hydroxyl group of the substrate, thereby generating the corresponding product. Alternatively, in the presence of oxidizing or reducing coenzymes, the recombinant host cell described in claim 7 can be used to reduce the carbonyl group or oxidize the hydroxyl group of the substrate through in vivo or in vitro reactions, thereby generating the corresponding product. The oxidative or reducing coenzyme is NADP(+) or NADPH; The substrate is N-demethylgalantamine, N-demethylnalidin, galantamine, or nalidin.

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