Reductase, nucleic acid fragment, recombinant expression vector, host cell and application thereof

Abstract

The invention relates to a reductase, a nucleic acid fragment, a recombinant expression vector, a host cell and application thereof. The amino acid sequence of the reductase is shown as SEQ ID NO: 1. According to the application, an enzyme for reducing 3-oxo-ionone into 3-oxo-ionol is found from tobacco, namely, a new reductase is provided, and the function of the enzyme in biosynthesis of related reductase genes is cloned and verified for the first time; in vitro, the recombinant protein can efficiently convert a 3-oxo-ionone substrate into a corresponding reduction product. The invention further provides a recombinant plasmid containing the reductase gene, the 3-oxo-ionol can be massively synthesized through a bioengineering method, meanwhile, the invention further provides application of the 3-oxo-ionol capable of being massively synthesized, more basic elements are provided for synthetic biology of natural products, and the application of the 3-oxo-ionol is expanded. In addition, guidance and basis are provided for rational design of the enzymes, and the method has good industrialization prospects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a reductase, a nucleic acid fragment, a recombinant expression vector, a host cell and application thereof. BACKGROUND

[0002] In tobacco, 3-oxo-ionone, as a core intermediate of terpene flavor precursors, connects the "glycoside storage form" and the "final flavor product" (such as megastigmatrienone, 3-oxo-alpha-ionone, etc.), and is an irreplaceable "bridge substance" in the formation chain of tobacco aroma. Compared with directly adding volatile flavor components, 3-oxo-ionone can be gradually converted during tobacco conditioning, storage and product combustion / heating stages due to its relatively stable chemical properties (containing hydroxyl functional groups, not easy to lose during processing), so as to realize "slow release and precise release" of aroma, and thus becomes a key direction of tobacco chemistry and process research.

[0003] At present, the preparation of tobacco flavoring and perfuming substances mainly relies on two ways of direct extraction of natural products or traditional chemical synthesis, but both have significant limitations: the extraction of natural products is limited by the scarcity of raw material resources, and it is difficult to realize large-scale and stable production; traditional chemical synthesis faces problems such as long route, harsh reaction conditions, and the risk of heavy metal pollution.

[0004] Therefore, the traditional technology still needs to be improved. SUMMARY

[0005] Based on this, the present application provides a reductase which can efficiently reduce the substrate to reduce 3-oxo-ionone to 3-oxo-ionol.

[0006] The specific scheme of the present application is as follows:

[0007] The first aspect of the present application provides a reductase, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0008] The second aspect of the present application provides a nucleic acid fragment, which encodes the above-mentioned reductase.

[0009] In some embodiments, the nucleotide sequence of the nucleic acid fragment is shown in SEQ ID NO: 2.

[0010] The third aspect of the present application provides a PCR primer pair, which can detect the above-mentioned nucleic acid fragment by PCR amplification reaction, and the nucleic acid fragment comprises an upstream primer with a nucleotide sequence shown in SEQ ID NO: 3 and a downstream primer with a nucleotide sequence shown in SEQ ID NO: 4.

[0011] The fourth aspect of the present application provides a recombinant expression vector comprising the above-mentioned nucleic acid fragment.

[0012] The fifth aspect of the present application provides a host cell expressing the reductase described above, or comprising the nucleic acid fragment described above or the recombinant expression vector described above.

[0013] The sixth aspect of the present application provides a preparation method of the reductase, comprising: after a host cell is transformed with a recombinant expression vector containing a nucleic acid fragment encoding the reductase, culturing the transformed host cell to prepare the reductase.

[0014] The seventh aspect of the present application provides a preparation method of 3-oxo-ionol, comprising the following steps:

[0015] mixing 3-oxo-ionone, a reducing coenzyme and the reductase to prepare 3-oxo-ionol.

[0016] In some embodiments, the concentration of the 3-oxo-ionone in the initial reaction system of the reaction is 0.01 mM to 100 mM;

[0017] and / or, the concentration of the reducing coenzyme in the initial reaction system of the reaction is 0.01 mM to 100 mM;

[0018] and / or, the temperature of the reaction is 16°C to 60°C, the time of the reaction is 0.1 h to 1 h, and the pH of the initial reaction system of the reaction is 5 to 9.

[0019] The eighth aspect of the present application further provides a preparation method of megastigmatrienone, comprising:

[0020] using the preparation method described above to prepare 3-oxo-ionol;

[0021] subjecting the 3-oxo-ionol to heat treatment or adding in a cigarette to dehydrate to obtain megastigmatrienone.

[0022] The present application discovers an enzyme that reduces 3-oxo-ionone to 3-oxo-ionol from tobacco, i.e., the present application provides a new reductase. In the research process, the applicant not only completes the cloning of the gene encoding the enzyme, but also determines its core role as a key reductase in the biosynthesis pathway through a functional verification experiment. The protein recombined and expressed from the gene can efficiently convert 3-oxo-ionone substrate and directionally generate the target reductive product.

[0023] On this basis, the present application further provides a recombinant expression vector containing the reductase gene: by means of the vector, the large-scale and low-cost synthesis of 3-oxo-ionol can be realized through biological engineering technologies such as microbial fermentation and enzyme catalysis, breaking through the efficiency bottleneck of traditional natural product extraction or chemical synthesis. BRIEF DESCRIPTION OF DRAWINGS

[0024] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The diagram shows the structure of the NSDR4-pET28a recombinant plasmid in Example 1;

[0026] Figure 2 This is a schematic diagram of the reaction process of 3-oxo-ionol in Example 1;

[0027] Figure 3 The 3-oxo-ionone in Example 1 1 H NMR ((700 MHz, DMSO-d6)) spectrum, where the horizontal axis represents chemical shift (δ) and the vertical axis represents signal intensity (relative integration area);

[0028] Figure 4 The 3-oxo-ionone in Example 1 13 C NMR (700 MHz, DMSO-d6) spectrum; where the horizontal axis represents chemical shift (δ) and the vertical axis represents signal intensity (relative integration area);

[0029] Figure 5 The image shows the chromatographic results of 3-oxo-ionol in Example 1. The horizontal axis represents retention time in minutes, and the vertical axis represents absorbance. Detailed Implementation

[0030] To facilitate understanding of this application, a more comprehensive description of the application will be provided below with reference to embodiments, and preferred embodiments of the application are given below. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are provided to provide a more thorough and complete understanding of the disclosure of this application. It should be understood that experimental methods in the following embodiments, unless specific conditions are specified, are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used reagents used in the embodiments are commercially available products.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0032] The terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element preceded by the phrase “comprising one…” does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. The indefinite articles “a” and “an” preceding an element or component in this application are not restrictive in terms of the quantity (i.e., the number of times) of the element or component. Therefore, “an” or “a” should be interpreted as including one or at least one, and singular elements or components also include plural forms, unless the quantity clearly refers only to the singular. “A plurality” means at least two, such as two, three, etc., unless otherwise expressly specified.

[0033] The terms "first," "second," and "third," etc., are used only for distinguishing descriptive purposes and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly specifying the importance or quantity of the indicated technical features. Moreover, "first," "second," etc., serve only a non-exhaustive enumeration purpose and should be understood not to constitute a closed limitation on quantity.

[0034] A "vector" is a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of the protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40).

[0035] "Host cell" refers to a cell that can be used to introduce the vector, including but not limited to animal cells such as Escherichia coli, fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0036] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the weights mentioned in the embodiments of this application can be well-known units of mass in the chemical industry, such as μg, mg, g, and kg.

[0037] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0038] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0039] One embodiment of this application provides a reductase, the amino acid sequence of which is shown in SEQ ID NO:1.

[0040] Specifically, such as SEQ ID The amino acid sequence shown in NO:1 is: MAEASNFLATQRIAVVTGANRGIGLEICRQLASKGIVVILTARDEKKGAEAIDILKECGLSDYVIFHKLDVTDPSTIAQLKDFIEVRFGKLDILVNSAAVLGVLMDKDVTIASPEQDLFVEKLKVATQTYDVAQECLKTNYYG AKQMIQELLPLLQFSDSPRIVNVSSIVGKLEHVRNEWAVGVLSDADNLTEERVDEVLNAFLQDLKDGLMETKNWSLIFPAYTLSKAAMNAYTRILAKKYPSLLINCVCPGYVKTDMTINCGKLSVEEGAESPVWLALLPKGGPSGKYFNRKEVSPF.

[0041] In addition, one embodiment of this application provides a nucleic acid fragment for encoding the reductase described above for secretion and expression in host cells.

[0042] Furthermore, the nucleotide sequence of the above nucleic acid fragment is as shown in SEQ ID NO:2, specifically: 5’-ATGGCAGAAGCTTCCAACTTCCTGGCAACCCAAAGGATTGCAGTTGTAACTGGAGCCAACAGAGGGATAGGACTAGAAATATGTAGACAGCTAGCTTCTAAGGGGATAGTGGTGATCTTAACAGCTAGAGATGAGAAGAAAGGGGCTGAAGCTATTGATATATTAAAGGAGTGTGGCCTATCTGATTATGTTATTTTTCATAAGCTTGATGTGACTGACCCTTCTACTATTGCACAACTCAAAGATTTCATCGAGGTCAGATTTGGCAAGCTTGATATCTTGGTGAATAGTGCAGCAGTTCTTGGAGTGCTTATGGATAAAGATGTTACAATTGCAAGTCCAGAACAAGACCTATTCGTTGAGAAGCTAAAGGTAGCAACTCAAACTTATGATGTAGCTCAAGAATGTCTAAAAACAAACTACTATGGGGCAAAACAGATGATTCAAGAATTATTACCTCTACTTCAATTTTCTGATTCACCTCGTATTGTCAATGTCTCCTCCATTGTTGGAAAGTTAGAGCATGTACGCAATGAATGGGCAGTAGGAGTGTTAAGTGATGCTGATAATCTAACAGAAGAGAGAGTAGATGAGGTTTTGAATGCTTTTTTGCAAGATTTGAAGGATGGTTTGATGGAAACTAAAAACTGGTCTCTGATTTTTCCTGCATATACACTATCAAAAGCAGCAATGAATGCTTACACAAGAATATTAGCCAAAAAATACCCAAGTCTTCTCATCAACTGTGTTTGTCCAGGCTATGTCAAAACTGATATGACTATCAATTGTGGCAAATTGAGTGTTGAAGAGGGTGCTGAAAGTCCTGTTTGGCTTGCTCTTTTACCTAAGGGAGGCCCATCAGGAAAATACTTCAATAGGAAAGAGGTGTCACCTTTCTAA-3’。

[0043] In some of these embodiments, the aforementioned nucleic acid fragments can be prepared by conventional chemical synthesis or other methods.

[0044] In some embodiments, the aforementioned nucleic acid fragments may be added to a recombinant expression vector to mediate the secretory expression of reductase.

[0045] One embodiment of this application provides a PCR primer pair capable of amplifying the aforementioned nucleic acid fragments.

[0046] As is understandable, a PCR primer pair consists of two artificially synthesized oligonucleotide sequences. One primer is complementary to one DNA template strand at one end of the target gene, and the other primer is complementary to the other DNA template strand at the other end of the target gene. In PCR (polymerase chain reaction) technology, given a known nucleotide sequence of the target gene, primers are synthesized based on this sequence. Using PCR amplification technology, the target gene DNA is denatured by heat and breaks down into single strands. The primers bind to the corresponding complementary sequences on the single strands. Then, extension is performed under the action of a thermostable DNA polymerase. This cycle is repeated, and the product obtained after extension can also bind to the primers.

[0047] In one specific example, the PCR primer pair described above includes an upstream primer with the nucleotide sequence shown in SEQ ID NO:3 and a downstream primer with the nucleotide sequence shown in SEQ ID NO:4. It is understood that PCR primer pairs capable of amplifying the above nucleic acid fragments are not limited to these, and PCR primer pairs with other sequences can be designed according to design rules.

[0048] Further, the nucleotide sequence shown in SEQ ID NO:3 is specifically: 5'-CAGCAAATGGGTCGCGGATCCATGGCAGAAGCTTCCAACTTCCTGGC-3'; the nucleotide sequence shown in SEQ ID NO:4 is specifically: 5'-CAAGCTTGTCGACGGAGCTCTTAGAAAGGTGACACCTCTTTCCTATTGAAGTATTTT-3'.

[0049] In addition, one embodiment of this application provides a recombinant expression vector containing the above-mentioned nucleic acid fragment encoding the reductase.

[0050] In one of the optional specific examples, the vector used to express the reductase is pET28a. The reductase expression vector is synthesized and constructed, the vector is double-digested with restriction endonucleases NdeI and SacI, and then gel electrophoresis is performed to recover the target fragment. The target sequence of the reductase is cloned into the restriction sites of NdeI and SacI, and the expression vector containing the reductase is transformed into host cells to express the target protein.

[0051] It can be understood that the recombinant expression vectors mentioned above are prokaryotic expression vectors.

[0052] In a specific example, the recombinant expression vector described above is E. coli DH5α. It is understood that the specific type of vector is not limited to this and can be selected according to specific needs.

[0053] This application further provides a recombinant expression vector containing the reductase gene: with the help of this vector, 3-oxo-ionol can be synthesized on a large scale and at low cost through bioengineering technologies such as microbial fermentation and enzyme catalysis, breaking through the efficiency bottleneck of traditional natural product extraction or chemical synthesis.

[0054] In addition, one embodiment of this application provides a host cell whose genome contains the above-mentioned recombinant expression vector, which can promote the secretion and expression of reductase.

[0055] In one embodiment, the host cell is *Escherichia coli*. Of course, in other embodiments, the host cell is not limited to *E. coli* and can be selected as needed.

[0056] Another embodiment of this application also provides a method for preparing the above-mentioned reductase, wherein the above-mentioned host cells are cultured to prepare the above-mentioned reductase.

[0057] Specifically, this includes transforming a recombinant expression vector containing a nucleic acid fragment encoding the aforementioned reductase into a host cell, culturing the transformed host cell, and preparing the reductase.

[0058] After culturing transfected host cells for a period of time, the host cells or the culture medium containing the host cells were collected to prepare a primary product containing the reductase. The primary product was then purified to prepare the purified reductase. The results showed that the reductase synthesized in this application can efficiently convert 3-oxo-ionone substrates and directionally generate the target reduced product.

[0059] One embodiment of this application also provides the use of the above-mentioned reductase, the above-mentioned nucleic acid fragment, the above-mentioned recombinant expression vector, or the above-mentioned host cell in the preparation of 3-oxo-ionol.

[0060] Another embodiment of this application provides a method for preparing 3-oxo-ionol, comprising the following steps: mixing 3-oxo-ionone, a reducing coenzyme and the above-mentioned reducing enzyme to prepare 3-oxo-ionol.

[0061] In some embodiments, the concentration of the 3-oxo-ionone in the above reaction system is 0.01 mM to 100 mM. For example, the concentration can be 0.01 mM, 0.1 mM, 1 mM, 3 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM or 100 mM. In some examples, it can be any two of these point values ​​as the end values, and the same applies below.

[0062] In a specific example, the concentration of the 3-oxo-ionone in the above reaction system is 3 mM.

[0063] In some embodiments, the concentration of the reductant coenzyme in the reaction system is 0.01 mM to 100 mM. For example, the concentration can be 0.01 mM, 0.1 mM, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM.

[0064] In a specific example, the concentration of the aforementioned reducing coenzyme in the above reaction system is 5 mM.

[0065] In one specific example, the aforementioned reducing coenzyme includes reduced nicotinamide adenine dinucleotide phosphate (NADPH).

[0066] In some embodiments, the concentration of the reductase in the reaction system is 0.1 mg / L to 100 mg / L. For example, the concentration can be 0.1 mg / L, 1 mg / L, 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, 25 mg / L, 30 mg / L, 35 mg / L, 40 mg / L, 45 mg / L, 50 mg / L, 55 mg / L, 60 mg / L, 65 mg / L, 70 mg / L, 75 mg / L, 80 mg / L, 85 mg / L, 90 mg / L, 95 mg / L, or 100 mg / L.

[0067] In one specific example, the concentration of the reductase in the above reaction system is 50 mg / L.

[0068] In some embodiments, the reaction temperature is 16°C to 60°C, the reaction time is 0.1h to 1h, and the pH is 5 to 9. For example, the temperature can be 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, 42°C, 44°C, 46°C, 48°C, 50°C, 52°C, 54°C, 56°C, 58°C, or 60°C; the time can be 0.1h, 0.5h, or 1h; and the pH can be 5, 6, 7, 8, or 9.

[0069] In a specific example, the reaction temperature was 30°C, the reaction time was 1 hour, and the pH was 7.5.

[0070] This application has discovered an enzyme in tobacco that reduces 3-oxo-ionone to 3-oxo-ionol, that is, this application provides a new reductase. In the research process, the applicant not only completed the cloning of the gene encoding this enzyme, but also clarified its core role as a key reductase in the biosynthetic pathway through functional verification experiments. The protein recombinantly expressed by this gene can efficiently convert 3-oxo-ionone substrates and directionally generate the target reduction product.

[0071] Based on this, this application further provides a recombinant expression vector containing the reductase gene: with the help of this vector, 3-oxo-ionol can be synthesized on a large scale and at low cost through bioengineering technologies such as microbial fermentation and enzyme catalysis, breaking through the efficiency bottleneck of traditional natural product extraction or chemical synthesis.

[0072] Another embodiment of this application provides a method for preparing megalotrienone, comprising: heat-treating the above-mentioned 3-oxo-ionol or adding it to cigarettes to dehydrate it, thereby obtaining megalotrienone.

[0073] In some embodiments, the temperature of the heat treatment is 110°C to 210°C.

[0074] Specifically, in cigarette manufacturing, the above-mentioned 3-oxo-ionol is dissolved in ethanol solvent as a cigarette flavoring.

[0075] In one specific example, the amount of 3-oxo-ionol added is 0.1% to 0.01% based on the weight of the tobacco. During cigarette smoking, 3-oxo-ionol is heated and dehydrated to form megazotrinone.

[0076] This application expands the application scenarios of the above-mentioned technology system, which can be used for the preparation of megastigmatrienone, providing a new path for the industrial production of tobacco aroma substances and natural active terpenoids, and providing important theoretical basis and experimental reference for the rational design of such enzymes, with good industrialization prospects.

[0077] To make the objectives, technical solutions, and advantages of this application clearer and more concise, the following specific embodiments are used for illustration, but this application is by no means limited to these embodiments. The embodiments described below are merely preferred embodiments of this application and can be used to describe this application, but should not be construed as limiting the scope of this application. It should be noted that any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application. Experimental methods not specified with specific conditions in the embodiments are implemented according to conventional conditions, such as those described in documents, books, or methods recommended by the manufacturer.

[0078] The plasmid pET28a used in the examples was preserved in our laboratory. The restriction enzymes NdeI and SacI, as well as the ligase, were purchased from Thermo Fisher Scientific. The competent cell preparation kit and plasmid purification kit were also purchased from Thermo Fisher Scientific. The E. coli DH5α strain and E. coli BL(21) strain used were preserved in our laboratory. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) was purchased from Beijing Cooler Master Technology Co., Ltd. The substrate 3-oxo-ionone was synthesized in our laboratory.

[0079] Example 1

[0080] 1. Construction of recombinant Escherichia coli reductase engineered bacteria

[0081] (1) The NSDR4 gene was obtained by amplifying the cDNA of tobacco leaves using NSDR4-specific primers (NSDR4F and NSDR4R). Specifically, the nucleotide sequences of the specific primers NSDR4F and NSDR4R are as follows: 5'-CAGCAAATGGGTCGCGGATCCATGGCAGAAGCTTCCAACTTCCTGGC-3'; and 5'-CAAGCTTGTCGACGGAGCTCTTAGAAAGGTGACACCTCTTTCCTATTGAAGTATTTT-3'.

[0082] (2) The NSDR4 fragment obtained by the above amplification was homologously recombined with the pET28a plasmid linearized by NdeI and SacI; the recombinant product was transformed into E. coli DH5α and screened by LB solid medium containing 50 μg / mL Kan (specific formula: 10 g / L tryptone, 5 g / L yeast extract powder, 10 g / L sodium chloride, pH 7.0-7.4).

[0083] (3) The recombinant plasmid NSDR4-pET28a obtained Figure 1 The NSDR4-pET28a recombinant plasmid was transformed into E. coli BL(21) to obtain a protein expression strain. The specific transformation steps were as follows: 100 ng of plasmid was added to E. coli BL(21) competent cells, placed on ice for 30 min, heat-treated at 42℃ for 90 s, placed on ice for 3 min, 900 μL of LB liquid medium was added, and cultured at 37℃ for 1 h. The culture was then spread on LB solid medium (containing 50 μg / mL) and cultured at 37℃ for 16 h. Single clones were selected for PCR verification.

[0084] PCR verification: Universal primers were T7 and T7t. T7 (SEQ ID NO:5): TAATACGACTCACTATAGGG; T7t (SEQ ID NO:6): GCTAGTATTGCTCAGCGG. The PCR amplification program was: 95℃ for 5 min; 95℃ for 30 s, 55℃ for 15 s, 72℃ for 1 min, 30 cycles, 72℃ for 5 min.

[0085] 2. Purification of reductase

[0086] (1) The protein expression strain obtained above was inoculated into LB medium containing Kan and cultured overnight at 37°C; then transferred to 2 L of LB liquid medium containing Kan at a ratio of 1% and incubated at 37°C until the bacterial culture OD. 600 = 0.6-0.8 and placed on ice to cool; add IPTG to a final concentration of 0.2 mM and induce overnight at 16°C for 18 h.

[0087] (2) Collect the above bacterial cells and resuspend them in Buffer A (20 mM imidazole, 50 mM Tris-HCl, 300 mM NaCl, 10% glycerol, pH=8.0) buffer. Sonicate on ice for 5 s, pause for 30 s, for a total of 4 min. Centrifuge the lysate at 24000 rpm for 2 h.

[0088] (3) Collect the supernatant, filter it through a 0.22 μm filter membrane, and load the filtrate onto a protein purification His column. Elute with 20 mL of Buffer A, and then elute with 20 mL of 250 mM imidazole buffer (Buffer A and Buffer B each 50%) (Buffer B: 500 mM imidazole, 50 mM Tris-HCl, 300 mM NaCl, 10% glycerol, pH=8.0). Collect the protein with 280 nm absorption. Detect the purification effect by SDS-PAGE electrophoresis.

[0089] (4) The collected liquid was placed in a 10 kDa molecular sieve to concentrate the protein, centrifuged at 4000 rpm, added 500 μL of storage solution (100 mM NaH2PO4 / Na2HPO4, 10% glycerol, pH=7.0), centrifuged at 4000 rpm, and then frozen in liquid nitrogen and stored at -80℃.

[0090] 3. Enzymatic activity detection and analysis of NSDR4-pET28a recombinant protein

[0091] Experimental group: In a 100 μL system, 50 mM Tris-HCl pH=7.5, 1 mM substrate (3-oxo-ionone), 1 mM NADPH, 50 μg NSDR4 enzyme, the reaction was carried out at 30℃ for 1 h. The reaction process is as follows. Figure 2 As shown, an equal volume of acetonitrile was added to stop the reaction, and the supernatant was collected by centrifugation for LC analysis.

[0092] Control group (CK): No enzyme was added, and all other conditions and parameters were the same as those of the experimental group.

[0093] The detection method was as follows: the chromatographic column was YMC-Triart C18 (ID 4.6×250 mm), the column temperature was 30℃, the flow rate was 1mL / min, 10-100% acetonitrile was used for 0-20 min, 100% acetonitrile for 20-24 min, and 10% acetonitrile for 24-28 min.

[0094] The NSDR4 enzyme was reacted with the substrate 3-oxo-ionone. The experimental group NSDR4 showed an additional product peak compared to the control group (CK), with retention times of 13.3 min and 13.5 min under analytical conditions, consistent with the elution time of the standard 3-oxo-ionone. The results are as follows: Figures 3-5 As shown, 3-oxo-ionone HE 1H NMR (700 MHz, DMSO-d6) δ 6.80 (1H,dd, J=15.8, 9.8), 6.15 (1H, d, J=15.8), 5.90 (1H, s), 3.32 (1H, s), 2.87 (1H,d, J=9.8), 2.25 (3H, s), 2.01 (1H, d, J=16.7), 1.86 (3H, s), 0.98 (3H, s), 0.92 (3H, s). 13 C NMR (175 MHz, DMSO-d6) δ 198.0, 197.6, 159.8, 144.7, 134.0, 125.8, 54.5, 47.0, 36.0, 27.3, 26.9, 26.8, 22.8, where Figure 3 For 3-oxo-ionone 1 HNMR (700 MHz, DMSO-d6) spectrum, Figure 4 For 3-oxo-ionone 13 C NMR (700 MHz, DMSO-d6) spectrum, Figure 5 The chromatogram of 3-oxo-ionol is shown above. Figure 5 It was found that the recombinase NSDR4-pET28a has reducing properties for 3-oxo-ionone and a conversion efficiency of 70%.

[0095] In summary, the technical solution of this application can realize the enzymatic reduction of 3-oxo-ionone to 3-oxo-ionol for the preparation of megalotrienone, with high conversion rate and purity, and high preparation efficiency.

[0096] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0097] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of this patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A reductase, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

1.

2. A nucleic acid fragment, characterized in that, Its encoding is the reductase as described in claim 1.

3. The nucleic acid fragment according to claim 2, characterized in that, The nucleotide sequence of the nucleic acid fragment is shown in SEQ ID NO:

2.

4. A PCR primer pair, characterized in that, The PCR primer pair is capable of amplifying the nucleic acid fragment of claim 2 or 3, wherein the nucleic acid fragment comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO:3 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO:

4.

5. A recombinant expression vector, characterized in that, Includes the nucleic acid fragments described in claim 2 or 3.

6. A host cell, characterized in that, It expresses the reductase of claim 1, or it comprises the nucleic acid fragment of any one of claims 2 to 3 or the recombinant expression vector of claim 5.

7. A method for preparing the reductase according to claim 1, characterized in that, include: The reductase is prepared by culturing the host cells according to claim 6.

8. A method for preparing 3-oxo-ionol, characterized in that, Includes the following steps: 3-O-ionone, a reducing coenzyme, and the reducing enzyme described in claim 1 are mixed and reacted to prepare 3-oxo-ionol.

9. The method for preparing 3-oxo-ionol according to claim 8, characterized in that, The concentration of the 3-oxo-ionone in the initial reaction system is 0.01 mM to 100 mM; And / or, the concentration of the reducing coenzyme in the initial reaction system is 0.01 mM to 100 mM; And / or, the reaction temperature is 16℃~60℃, and the reaction time is 0.1h~1h; And / or, the initial pH of the reaction system is 5-9.

10. A method for preparing megastigmatrienone, characterized in that, include: 3-Oxo-ionol was prepared by the method described in any one of claims 8 to 9; The 3-oxo-ionol was subjected to heat treatment or added to cigarettes for dehydration to obtain megastigmatrienone.

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