Use of geranylgeranyl pyrophosphate reductase in the preparation of tocopherols

Abstract

The application relates to an application of a geranylgeranyl pyrophosphate reductase in preparation of tocopherol, wherein a nucleotide sequence coding the geranylgeranyl pyrophosphate reductase is shown as SEQ ID No. 11, and the application comprises the following steps: under the assistance of coenzyme iron-oxidation-reduction protein and iron-oxygen-reduction protein-NADP+ reductase, the geranylgeranyl pyrophosphate reductase catalyzes the reduction of delta-tocotrienol to generate delta-tocopherol. The geranylgeranyl pyrophosphate reductase has the function of reducing the unsaturated isoprene side chain of tocotrienol, can catalyze the reduction of tocotrienol to generate tocopherol, does not need to depend on the geranyl pyrophosphate precursor in the existing tocopherol biosynthesis pathway, significantly improves the tocopherol synthesis efficiency, and provides a new strategy for the biosynthesis of tocopherol and other tocopherols.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to the application of a geraniol-geraniol pyrophosphate reductase in the preparation of tocopherol. Background Technology

[0002] Vitamin E is a fat-soluble compound with antioxidant activity and is an essential vitamin for maintaining metabolism. It is widely used in food, medicine, and cosmetics, with a huge market demand. Vitamin E consists of a polar aromatic head derived from the shikimic acid pathway and a lipophilic isoprene side chain derived from the terpene synthesis pathway. Based on the saturation of the side chain and the number and position of methyl groups on the aromatic ring, it is classified into α-tocotrienol, β-tocotrienol, γ-tocotrienol, δ-tocotrienol, and α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol. In humans and other mammals, tocopherol has higher activity and can be preferentially transported and absorbed.

[0003] Natural vitamin E is produced only in plants and photosynthetic algae. The synthesis of tocotrienols and tocopherols both begin with two precursors: 4-hydroxyphenylpyruvate and geranyl-geranyl pyrophosphate (GGPP). 4-hydroxyphenylpyruvate is catalyzed by p-hydroxyphenylpyruvate dioxygenase (HPPD) to produce homogentisic acid. GGPP and homogentisic acid are then catalyzed by homogentisic acid chlorophyll transferase (HPT) to produce 2-methyl-6-geranyl-geranyl-benzoquinone (MGGBQ), which is further catalyzed by tocopherol cyclase (TC) to produce δ-tocotrienol. Similarly, GGPP is catalyzed by geranyl-geranyl pyrophosphate reductase to produce phytophosphate (PDP). Homogentisic acid and PDP are then catalyzed by HPT to produce 2-methyl-6-phytophosphate-benzoquinone (MPBQ), which is further catalyzed by TC to produce δ-tocopherol.

[0004] However, current research on geranyl-geranyl pyrophosphate reductase is limited, and research on the heterologous synthesis of δ-tocopherol and other tocopherol configurations via microorganisms is scarce and developing slowly. The core bottleneck lies in the insufficient supply of PDP precursors in the aforementioned synthetic pathways, resulting in low tocopherol synthesis efficiency and difficulty in meeting the needs of industrial production. Therefore, developing efficient new synthetic pathways for tocopherol is key to solving the problem of large-scale tocopherol production. Summary of the Invention

[0005] This invention aims to at least partially solve one of the technical problems in related technologies. Therefore, the objective of this invention is to provide an application of geranyl-geranyl pyrophosphate reductase in the preparation of tocopherol. This geranyl-geranyl pyrophosphate reductase possesses isoprene-reducing activity and can catalyze the reduction of unsaturated side chains of tocotrienols to tocopherol. Based on this, a new tocopherol synthesis pathway bypassing phytophosphate precursors is constructed, significantly improving the efficiency of tocopherol synthesis.

[0006] Therefore, in one aspect of the present invention, an application of geranylgeranyl pyrophosphate reductase in the preparation of tocopherol is proposed, wherein the nucleotide sequence encoding the geranylgeranyl pyrophosphate reductase is shown in SEQ ID No. 11, and the application includes: with the assistance of coenzyme ferrooxidoreductase and ferroredoxin-NADP+ reductase, the geranylgeranyl pyrophosphate reductase catalyzes the reduction of δ-tocotrienol to δ-tocopherol.

[0007] The application of geraniol-geraniol pyrophosphate reductase according to the present invention in the preparation of tocopherol reveals for the first time that the geraniol-geraniol pyrophosphate reductase encoded by SEQ ID No. 11 has the activity of reducing the unsaturated isoprene side chain of δ-tocotrienol. This breaks the perception that the enzyme can only recognize geraniol-geraniol pyrophosphate (GGPP) as a natural substrate. It can directly convert δ-tocotrienol into δ-tocopherol without relying on the phytophosphate precursor in the existing tocopherol biosynthesis pathway, significantly improving the efficiency of tocopherol synthesis. This provides key functional enzyme support for the microbial synthesis of tocopherol and opens up a new metabolic node.

[0008] In a second aspect, the present invention provides an enzyme composition for preparing tocopherol, the enzyme composition comprising geraniol geraniol pyrophosphate reductase, ferric oxide reductase and ferric oxide reductase-NADP+ reductase. The nucleotide sequence encoding the geranylgeranyl pyrophosphate reductase is shown in SEQ ID No. 11; The enzyme composition is used to catalyze the reduction of δ-tocotrienol to δ-tocopherol, and nicotinamide adenine dinucleotide phosphate is added to the catalytic system as an electron donor.

[0009] The enzyme composition of the present invention can directly synthesize δ-tocopherol from δ-tocotrienol substrates, bypassing the existing plant-based pyrophosphate precursor synthesis pathway. By combining with nicotinamide adenine dinucleotide phosphate (NADPH) electron donor, the synthesis efficiency is significantly improved, providing a complete functional module for microbial synthesis of tocopherol.

[0010] In a third aspect, the present invention provides a method for constructing an engineered strain of *Escherichia coli* that produces tocopherol, comprising: constructing a recombinant plasmid expressing the gerany-gerany pyrophosphate reductase BchP gene, the ferrooxidase FdxA gene, and the ferroredoxin-NADP+ reductase Fdr gene; wherein the nucleotide sequence of the gerany-gerany pyrophosphate reductase is shown in SEQ ID No. 11. The recombinant plasmid was electroporated into Escherichia coli to obtain an engineered Escherichia coli strain that produces tocopherol.

[0011] According to the construction method of the present invention, Escherichia coli is used as the expression chassis, which has a clear genetic background, low culture cost, and high expression efficiency. It can achieve soluble co-expression of three functional enzymes. The constructed engineered strain can be directly used for whole-cell catalysis or preparation of crude enzyme solution without complicated protein purification steps, which greatly reduces the cost of industrial application. The three functional genes are co-expressed on the same recombinant plasmid, eliminating the need to construct a multi-plasmid system. It has high genetic stability and is suitable for large-scale fermentation production.

[0012] In a fourth aspect, the present invention provides a method for preparing tocopherol, comprising: Provide the above-described enzyme composition, or a lysate of an engineered Escherichia coli strain constructed using the above-described construction method; Add tocotrienol substrate and nicotinamide adenine dinucleotide phosphate to the reaction system and react at 30-37℃ for 12-24 hours to obtain the reaction product; Tocopherol was obtained by separating and purifying the reaction product.

[0013] The method for preparing tocopherol according to the present invention has mild reaction conditions and can be coupled with the fermentation production process of tocotrienols to achieve a fully biological preparation from raw materials such as inexpensive carbon sources to products. The natural properties of the products meet the application requirements of the food and pharmaceutical fields, are environmentally friendly, and are suitable for large-scale industrial production.

[0014] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0015] Figure 1 This is the tocopherol synthesis pathway according to an embodiment of the present invention; wherein, BchP: gerany-gerany-pyrophosphate reductase; FdxA: iron oxide redox protein (Fd ox ) / Reduced iron redox protein (Fd red Fdr: ferroredoxin-NADP+ reductase; Figure 2 To provide an embodiment of the present invention, spheroidal red bacteria ( R. sphaeroides BchP protein or BchP-FdrxA-Fdr combined protein in Escherichia coli (E. coli) E. coliHeterologous expression was performed in BL21 (DE3) to achieve the synthesis of δ-tocopherol (δ-T) from δ-tocotrienol (δ-T3); where Ec-Control: E. coli expressing the empty vector; Ec-BchP: E. coli expressing the pRSF-BchP vector; Ec-BchP-FdrxA-Fdr: E. coli expressing the pRSF-BchP-FdrxA-Fdr vector; the experiments were carried out using cell lysate as a biocatalyst, and all experiments were performed in triplicate. Only representative chromatograms (retention times, range 396-435 m / z) are shown, and * indicates the target tocopherol product. Detailed Implementation

[0016] The technical solution of the present invention is illustrated below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or defining the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0017] To better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

[0018] The test materials used in this invention are all commercially available products and can be purchased on the market; unless otherwise specified, the experiments involved are all conventional experimental methods.

[0019] Materials used: Escherichia coli BL21 (DE3) and TOP10 were commercially available. Escherichia coli BL21 (DE3) was used for the expression of all genes in this application, and TOP10 was used for vector construction. The E. coli expression vector pRSFDuet-1 was obtained from Novagen. Phusion high-fidelity DNA polymerase, T4 DNA ligase, kanamycin, etc., were purchased from Xiamen Lulong Biotechnology Development Co., Ltd. Plasmid extraction kit, DNA purification kit, gel extraction kit, and genomic DNA extraction kit were purchased from Shanghai Sangon Biotech Co., Ltd.; homologous recombination kit was purchased from Wuhan Aibotek Biotechnology Co., Ltd. Primer synthesis was performed by Shanghai Sangon Biotech Co., Ltd.

[0020] The composition of LB medium is: 10 g·L -1 Peptone, 5 g·L -1 Yeast powder, 10 g·L -1 NaCl, balance double-distilled water, autoclave at 121℃ for 30 min, add 15 g·L⁻¹ as needed. -1 Agar powder is used to make a solid culture medium.

[0021] The composition of TB culture medium is: 12 g L -1 Peptone, 24 g / L -1 Yeast powder, 2.31 g / L -1 KH2PO4, 12.54 g / L -1 K2HPO4, 0.4% glycerol, balance double-distilled water, autoclaved at 121℃ for 30 min.

[0022] δ-Tocotrienol and δ-Tocopherol standards were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0023] To determine the product's composition, δ-tocotrienol or δ-tocopherol standards were dissolved in acetone to prepare standard solutions of a specific concentration. The product extract and standards were then subjected to high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) using a Shimadzu C18 column (4.6 × 250 mm, 5 μm), a photodiode array detector (operating wavelength 280 nm), and a Q Exactive mass spectrometer equipped with a heated electrospray ionization interface and an electrospray ionization (ESI) device. The mobile phase was pure water (A) and acetonitrile (B), using a gradient elution program at a flow rate of 0.3 mL / min. -1 The column temperature was 40℃, and the injection volume was 10 μL. The linear gradient was applied from 50% A / 50% B to 0% A / 100% B for 0-5 min; maintained at 0% A / 100% B for 5-15 min; and from 15-15.5 min, the linear gradient was applied from 0% A / 100% B to 50% A / 50% B for 15.5-20 min; maintaining at 50% A / 50% B for 15.5-20 min. Ion source parameters were optimized: positive mode spray voltage 3.5 kV, negative mode spray voltage 3.0 kV. Other instrument parameters were: capillary temperature 320℃, auxiliary gas heating temperature 350℃, sheath gas pressure 30 arb, auxiliary gas pressure 5 arb, purge gas pressure 1 arb, and S-lens RF voltage 60 V. The Q Exactive detector was used in full scan mode, with a Fourier transform (FT) resolution set to 70000. The target ion number was 1×10⁻⁶ for automatic gain control (AGC). 6The maximum injection time is 50 ms. The mass-to-charge ratio (m / z) of mass spectrometry fragment scans of sample extracts and standards ranges from 100 to 1000.

[0024] Table 1 Primers used for PCR amplification

[0025] Table 2. DNA sequences of plasmids

[0026] The biosynthetic pathway of tocopherol in this invention is shown in the following embodiments. Figure 1 : Ferroreductin-NADP+ reductase is responsible for collecting electrons from NADPH and transferring them to oxidized ferroreductin to convert it into reduced ferroreductin. Then, the reduced ferroreductin is responsible for transferring electrons to geraniol-geraniol pyrophosphate reductase. Finally, geraniol-geraniol pyrophosphate reductase uses electrons to reduce the unsaturated isoprene side chain of tocotrienol, thereby converting it into tocopherol.

[0027] In embodiments of the present invention, a novel key tocotrienol reductase (geranylgeranyl pyrophosphate reductase BchP) was identified in *Rhodotorula spheroides*, which is derived from... bchP Encoding; and based on coenzyme ferrooxidase FdxA and ferroredoxin-NADP+ reductase Fdr, a new tocopherol synthesis pathway is formed; the cofactor NADPH is also added to the reaction system to provide an electron donor for the enzymatic reaction of BchP.

[0028] More specifically, based on this gerany-gerany pyrophosphate reductase BchP, the existing synthetic pathway for tocopherol production from plant-based pyrophosphate precursors can be replaced by the reduction of tocotrienols to tocopherols. This improves the production efficiency of tocopherols and provides a new strategy for the industrial production of natural tocopherols. Combined with the tocotrienol biosynthetic pathway, this holds promise as an alternative to the extraction of natural tocopherols from plants, with broad prospects for industrial application.

[0029] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0030] Example 1: Construction of Recombinant Escherichia coli 1. Constructing the pRSF-BchP plasmid Using the genome of *Rhodops spherulites* 2.4.1 (GenBank: CP000143.2) as a template, and primers of BchP-F2 (SEQ ID No. 9) and BchP-R2 (SEQ ID No. 10), the BchP gene fragment was amplified. The target band was verified by gel electrophoresis, and the purified DNA fragment was obtained using a gel extraction and purification kit. PCR amplification conditions: 98℃ for 3 min; 98℃ for 15 s, 64℃ for 60 s, 72℃ for 3 min, 30 cycles; 72℃ for 5 min, then hold at 4℃.

[0031] The BchP gene fragment was used Bam HI and Xho I enzyme digestion, followed by ligation into... Bam HI and Xho The expression vector pRSFDuet-1, which was double-digested with enzyme I, was ligated overnight at 16°C using T4 DNA ligase. The ligated product was transformed into E. coli Top10 competent cells, and single colonies were cultured. Using primers BchP-F2 (SEQ ID No. 9) and BchP-R2 (SEQ ID No. 10) as primers, positive single colonies were obtained by PCR verification. The recombinant plasmid was extracted, and the correct recombinant plasmid pRSF-BchP was obtained after sequencing. The nucleotide sequence is shown in SEQ ID No. 12.

[0032] 2. Constructing the pRSF-BchP-FdxA-Fdr plasmid Using the genome of *Rhodopseudomonas spheroidae* 2.4.1 as a template, the BchP gene fragment was amplified by PCR using primers BchP-F (SEQ ID No. 1) and BchP-R (SEQ ID No. 2). The FdxA gene fragment was amplified by PCR using primers FdxA-F (SEQ ID No. 3) and FdxA-R (SEQ ID No. 4). The Fdr gene fragment was amplified by PCR using primers Fdr-F (SEQ ID No. 5) and Fdr-R (SEQ ID No. 6). Using the vector pRSFDuet-1 as a template, the gene fragment of the vector backbone pRSF was amplified by PCR using primers pRSF-F (SEQ ID No. 7) and pRSF-R (SEQ ID No. 8). The target bands were validated by gel electrophoresis, and purified DNA fragments were obtained using a gel extraction and purification kit. PCR amplification conditions: 98℃ for 3 min; 98℃ for 15 s, 64℃ for 60 s, 72℃ for 3 min, 30 cycles; 72℃ for 5 min, hold at 4℃.

[0033] The BchP gene fragment, FdxA gene fragment, Fdr gene fragment, and the gene fragment of the vector backbone pRSF were seamlessly cloned and ligated using a homologous recombination kit. The ligated product was transformed into E. coli Top10 competent cells, and single colonies were selected for culture. Using BchP-F (nucleotide sequence SEQ ID No. 1) and Fdr-R (nucleotide sequence SEQ ID No. 6) as primers, positive single colonies were obtained by PCR verification. The recombinant plasmid was extracted, and the correct recombinant plasmid pRSF-BchP-FdxA-Fdr was obtained after sequencing. The nucleotide sequence is shown in SEQ ID No. 13.

[0034] 3. Obtaining recombinant strains of Escherichia coli The recombinant plasmids pRSF-BchP and pRSF-BchP-FdxA-Fdr, as well as the empty vector pRSFDuet-1, were transformed into Escherichia coli BL21 (DE3) using an electroporation method. The strains were screened in LB medium containing kanamycin to obtain positive recombinant strains Ec-BchP, Ec-BchP-FdxA-Fdr, and Ec-Control, respectively.

[0035] Example 2: Bioconversion of whole cell lysate to synthesize tocopherol To determine the presence of Rhodopsylum spheroidans bchPThe gene (nucleotide sequence shown in SEQ ID No. 11) encodes geraniol pyrophosphate reductase (Protein ID: ABA79450.1) for reducing the unsaturated isoprene side chain of tocotrienols, and the necessity of coenzymes: ferrooxidoreductin FdxA (Protein ID: ABA78585.1) and ferroreductin-NADP+ reductase Fdr (Protein ID: ABA78090.1).

[0036] The recombinant strains Ec-BchP, Ec-BchP-FdxA-Fdr, and Ec-Control prepared in Example 1 were inoculated into LB medium and cultured at 37°C for 12-16 hours to obtain seed culture. 1% of the fresh overnight seed culture was inoculated into 200 mL of TB medium and cultured at 37°C for approximately 2-3 hours. OD 600 Once the concentration reaches 0.4-0.6, 0.1 mM isopropyl-β-D-thiogalactoside (IPTG) is added, and protein expression is induced at 20°C for approximately 16-24 h. Subsequently, the bacterial cells are collected, washed twice with 50 mM Tris-HCl buffer (pH 8.0), resuspended in the same buffer, and then sonicated to prepare cell lysate.

[0037] The total volume of the biotransformation system was 1 mL, containing 0.1 mM δ-tocotrienol, 0.5 mM NADPH, cell lysis buffer biocatalyst equivalent to 10 g dry cell weight (CDW) / L, and 50 mM Tris-HCl buffer (pH 8.0) to make up the volume. The reaction was carried out at 30 ℃ and 250 rpm for 24 hours.

[0038] The reaction mixture was extracted with 500 mL of acetone and transferred to a 2 mL centrifuge tube. The supernatant was collected by centrifugation, filtered, and then sampled into a liquid chromatography bottle. Qualitative analysis of the product was performed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). Results are as follows: Figure 2 As shown, cell lysates of both the control strain Ec-Control and the recombinant strain Ec-BchP did not react with δ-tocotrienol. However, cell lysates of the recombinant strain Ec-BchP-FdxA-Fdr did react with δ-tocotrienol and generate δ-tocopherol. These experimental results indicate that *Rhodops spheroidae*... bchP The gene-encoded geraniol-geraniol pyrophosphate reductase has a first-reported function of reducing the unsaturated isoprene side chain of tocotrienols, and requires the assistance of coenzyme ferrooxidoreductase FdxA and ferroreductin-NADP+ reductase.

[0039] In summary, embodiments of the present invention provide a novel biosynthetic pathway for tocopherol (e.g.) Figure 1 As shown, tocopherol is produced from the direct precursor triene tocopherol via gerany-gerany-pyrophosphate reductase, thus distinguishing it from existing synthetic pathways that produce tocopherol from phytophosphate precursors.

[0040] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. The illustrative expressions of the above terms in this specification should not be construed as necessarily referring to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

[0041] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. The application of a geranyl-geranyl pyrophosphate reductase in the preparation of tocopherol, characterized in that, The nucleotide sequence encoding the geraniol-geraniol pyrophosphate reductase is shown in SEQ ID No.

11. The application includes: catalyzing the reduction of δ-tocotrienol to δ-tocopherol with the assistance of coenzyme ferrooxidase and ferroredoxin-NADP+ reductase.

2. The application according to claim 1, characterized in that, Nicotinamide adenine dinucleotide phosphate was added as an electron donor when catalyzing the δ-tocotrienol.

3. The application as described in claim 1, characterized in that, The geranylgeranyl pyrophosphate reductase is derived from *Rhodotorula spheroidae*. Rhodobacter sphaeroides 2.4.

1.

4. An enzyme composition for preparing tocopherol, characterized in that, The enzyme composition includes geraniol geraniol pyrophosphate reductase, ferric oxide reductase and ferric oxide-NADP+ reductase. The nucleotide sequence encoding the geranylgeranyl pyrophosphate reductase is shown in SEQ ID No. 11; The enzyme composition is used to catalyze the reduction of δ-tocotrienol to δ-tocopherol, and nicotinamide adenine dinucleotide phosphate is added to the catalytic system as an electron donor.

5. The enzyme composition according to claim 4, characterized in that, The ferroredoxin-NADP+ reductase collects electrons from the nicotinamide adenine dinucleotide phosphate and transfers them to the oxidized ferroredoxin, which is then converted into the reduced ferroredoxin. The reduced ferroredoxin then transfers electrons to the geranylgeranyl pyrophosphate reductase, which uses the electrons to reduce the unsaturated isoprene side chain of δ-tocotrienol, thereby converting it into δ-tocopherol.

6. A method for constructing an engineered strain of *Escherichia coli* that produces tocopherol, characterized in that, include: A recombinant plasmid expressing the gerany-gerany pyrophosphate reductase BchP gene, the ferrooxidase FdxA gene, and the ferroredoxin-NADP+ reductase Fdr gene was constructed; the nucleotide sequence of the gerany-gerany pyrophosphate reductase is shown in SEQ ID No.

11. The recombinant plasmid was electroporated into Escherichia coli to obtain an engineered Escherichia coli strain that produces tocopherol.

7. The construction method as described in claim 6, characterized in that, The nucleotide sequence of the recombinant plasmid is shown in SEQ ID No.

13.

8. The construction method as described in claim 6, characterized in that, Includes the following steps: Using the genome of Rhodops spheroidae 2.4.1 with accession number CP000143.2 as a template, and using BchP-F (nucleotide sequence SEQ ID No. 1) and BchP-R (nucleotide sequence SEQ ID No. 2) as primers, the gene fragment of gerany gerany pyrophosphate reductase BchP was amplified by PCR. Using the genome of Rhodops spheroidae 2.4.1 with accession number CP000143.2 as a template, and using FdxA-F (nucleotide sequence SEQ ID No. 3) and FdxA-R (nucleotide sequence SEQ ID No. 4) as primers, the gene fragment of the ferrooxidation protein FdxA was amplified by PCR. Using the genome of Rhodops spheroidae 2.4.1 with accession number CP000143.2 as a template, and using Fdr-F (nucleotide sequence SEQ ID No. 5) and Fdr-R (nucleotide sequence SEQ ID No. 6) as primers, the gene fragment of ferroredoxin-NADP+ reductase Fdr was amplified by PCR. Using the vector pRSFDuet-1 as a template, and using the nucleotide sequence pRSF-F shown in SEQ ID No. 7 and the nucleotide sequence pRSF-R shown in SEQ ID No. 8 as primers, the gene fragment of the vector backbone pRSF was obtained by PCR amplification. The recombinant plasmid was obtained by seamless cloning of the gene fragments of the gerany-gerany pyrophosphate reductase BchP, the ferric oxide-reductase FdxA, the ferric oxide-NADP+ reductase Fdr, and the vector backbone pRSF through homologous recombination. The recombinant plasmid was transformed into E. coli. Escherichia coli The engineered strain of Escherichia coli that produces tocopherol was obtained in BL21 (DE3).

9. A method for preparing tocopherol, characterized in that, include: Provide the enzyme composition of claim 4 or 5, or the lysate of an engineered strain of Escherichia coli constructed by any one of claims 6-8; Add tocotrienol substrate and nicotinamide adenine dinucleotide phosphate to the reaction system and react at 30-37℃ for 12-24 hours to obtain the reaction product; Tocopherol was obtained by separating and purifying the reaction product.

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