A method for biocatalytic conversion of β-ionol to prepare dihydro-β-ionone

Abstract

The present invention discloses a method for biocatalytic conversion of β-ionol to prepare dihydro-β-ionone, belonging to the technical field of bioengineering. In the present invention, β-ionol is used as a substrate, and short-chain alcohol dehydrogenase NaSDR and enoate reductase AaDBR1 are added for reaction. The reaction temperature is 25-60 °C, the pH is 4.0-11.0, and the reaction time is 0.5-3 h to obtain β-ionone. The present invention uses short-chain alcohol dehydrogenase and enoate reductase for cascade reaction, establishes a process for in vitro one-pot biopreparation of dihydro-β-ionone using β-ionol as a substrate, and realizes the regeneration and self-circulation of NADPH, thus creating a new method for highly efficient and low-cost biosynthesis of dihydro-β-ionone.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and particularly relates to a method for biocatalytic conversion of β-ionol to prepare dihydro-β-ionone. Background Art

[0002] Dihydro-β-ionone, a secondary metabolite derived from plants such as osmanthus, has a cyclic monoterpene structure and is commonly known as the "king of osmanthus". It is the main aroma compound with a mellow, sweet, and fresh cedar fragrance in osmanthus oil. The warm, woody, berry, and unique violet odor of dihydro-β-ionone makes it a precious and rare new fragrance. Currently, dihydro-β-ionone has successfully found its place in most high-end perfumes, cosmetics, and food flavor additives. In addition, dihydro-β-ionone also has activities such as anti-cancer, anti-inflammatory, and antibacterial, and has potential application prospects in the fields of healthcare and pharmaceuticals.

[0003] In recent years, the global market demand for dihydro-β-ionone is approximately 10 - 100 tons per year. However, dihydro-β-ionone derived from natural plants far from meets the market demand. Currently, dihydro-β-ionone is mainly obtained through chemical synthesis. However, the chemical synthesis process is not only complex but also environmentally unfriendly, resulting in the obtained product not being able to be sold as a natural product. Although dihydro-β-ionone prepared by the plant extraction method is a natural product, its yield is extremely low, and a large amount of plant materials are required, leading to an increase in the preparation cost. Thus, both of these methods limit the practical application of dihydro-β-ionone. With the rapid development of biotechnology, the production of aromatic compounds using biocatalysis and transformation technologies or synthetic biology means has attracted increasing attention and will become an effective means to replace plant extraction and chemical synthesis. Currently, the biosynthetic pathways of typical aroma substances such as linalool and geraniol have been elucidated, and high-level biosynthesis has been achieved. In terms of the biological preparation of dihydro-β-ionone, Zhao Lingguo et al. (Zhao Lingguo, Zhang Xuesong, Liao Shiyong, Tang Feng, Pei Jianjun, Application of Artemisia aldehyde double bond reductase DBR1 and its recombinant bacteria in the preparation of dihydro-β-ionone, authorization date: December 8, 2020, authorization patent number: ZL201710332991.7) have shown that the enoate reductase AaDBR1 in Artemisia annua has high selectivity for the hydrogenation of the 10,11-unsaturated double bond in β-ionone and can efficiently catalyze the conversion of β-ionone to dihydro-β-ionone. Therefore, based on the heterologous synthesis pathway of β-ionone in Escherichia coli, an enoate reductase was introduced, and a biosynthetic pathway for the de novo synthesis of dihydro-β-ionone was designed. However, the existing technology has the following technical bottlenecks: The CCD enzyme is an important rate-limiting enzyme in the process of converting β-carotene to β-ionone, and the activities and specificities of CCD enzymes from different sources vary greatly; Based on the heterologous synthesis pathway of β-ionone in Escherichia coli, the introduction of an enoate reductase will cause the imbalance of the strain's metabolic pathway, resulting in metabolic disorders, and the growth of the bacteria is significantly inhibited, no dihydro-β-ionone is produced, and at the same time, the yield of β-ionone is significantly reduced; During the de novo synthesis process, the spatial contact ability between β-ionone and the enoate reductase is limited, etc. Therefore, the de novo biosynthesis of dihydro-β-ionone is difficult, and it is difficult to achieve large-scale production in a short time.

[0004] Artificially designed enzyme cascade platforms have recently been developed as powerful systems for the biomanufacture of natural products. Enzyme reactions in different steps can be precisely manipulated in vitro to achieve efficient biosynthesis, which features high product yields, enhanced mass transfer, shorter reaction times, and stronger tolerance to toxic products. Currently, Qi et al. (Zhipeng Qi, Xinyi Tong, Kaixuan Ke, Xinyi Wang, Jianjun Pei, Su Bu, *Linguo Zhao * The de novo synthesis of dihydro-β-ionone from carotenoids by the coupled catalysis of carotenoid cleavage dioxygenase and enoate reductase was demonstrated in “De Novo Synthesis of Dihydro-β-ionone through Metabolic Engineering and Bacterium-Yeast Coculture, Journal of Agricultural and Food Chemistry, 2024, 72, 3066−3076”. However, the lack of excellent CCD enzymes and the hydrophobicity of carotenoids and their rapid degradation rate pose significant challenges in forming a homogeneous solution and storage, resulting in certain limitations in the synthesis of dihydro-β-ionone by this method. Therefore, it is particularly important to find substrates that can replace carotenoids. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section, as well as in the abstract and title of the present application, to avoid obscuring the purpose of this section, the abstract, and the title, but such simplifications or omissions shall not be used to limit the scope of the present invention.

[0006] In view of the above and / or problems existing in the prior art, the present invention is proposed.

[0007] One of the objectives of the present invention is to provide a method for biocatalytic conversion of β-ionol to prepare dihydro-β-ionone. By using short-chain alcohol dehydrogenase and enoate reductase for cascade reaction, a process for in vitro one-pot biopreparation of dihydro-β-ionone with β-ionol as the substrate is established, and the regeneration and self-circulation of NADPH are realized.

[0008] To solve the above technical problems, the present invention provides the following technical solution: A method for biocatalytic conversion of β-ionol to prepare dihydro-β-ionone, comprising:

[0009] Using β-ionol as the substrate, adding short-chain alcohol dehydrogenase NaSDR and enoate reductase AaDBR1 for reaction, with the reaction temperature being 25−60 °C, the pH being 4.0−11.0, and the reaction time being 0.5−3 h to obtain β-ionone.

[0010] As a preferred embodiment of the method for biocatalytic conversion of β-ionol to prepare dihydro-β-ionone in the present invention, wherein: the short-chain alcohol dehydrogenase NaSDR is derived from Novosphingobium aromaticivorans DSM 12444, and its amino acid sequence is shown in SEQ ID NO.1. The short-chain alcohol dehydrogenase NaSDR has the highest enzyme activity at 35 °C and pH 6.5. In the present invention, the method for obtaining the short-chain alcohol dehydrogenase NaSDR is not limited. The short-chain alcohol dehydrogenase NaSDR can be obtained by means such as culture, fermentation, separation and purification, or can also be obtained by gene cloning, expression, separation and purification.

[0011] As a preferred embodiment of the method for biocatalytic conversion of β-ionol to prepare dihydro-β-ionone in the present invention, wherein: the enoic acid reductase AaDBR1 is derived from Artemisia annua, and its amino acid sequence is shown in SEQ ID NO.2. The enoic acid reductase AaDBR1 has the highest enzyme activity at 45 °C and pH 6.5. In the present invention, the method for obtaining the enoic acid reductase AaDBR1 is not limited. The enoic acid reductase AaDBR1 can be obtained by means such as culture, fermentation, separation and purification, or can also be obtained by gene cloning, expression, separation and purification.

[0012] As a preferred embodiment of the method for biocatalytic conversion of β-ionol to prepare dihydro-β-ionone in the present invention, wherein: in the reaction system, the addition amount of the short-chain alcohol dehydrogenase NaSDR is 0.06 mg / mL to 0.35 mg / mL; the addition amount of the enoic acid reductase AaDBR1 is 0.08 mg / mL to 0.47 mg / mL.

[0013] As a preferred embodiment of the method for biocatalytic conversion of β-ionol to prepare dihydro-β-ionone in the present invention, wherein: the substrate concentration of β-ionol is 0.5 mM to 5.0 mM.

[0014] As a preferred embodiment of the method for biocatalytic conversion of β-ionol to prepare dihydro-β-ionone in the present invention, wherein: the reaction system further includes a cofactor, and the cofactor is NADP + , NAD + in combination with NADPH, NADP + in combination with NADPH, and the concentration of the cofactor is 0.125 to 2.0 mM.

[0015] As a preferred embodiment of the method for biocatalytic conversion of β-ionol to prepare dihydro-β-ionone in the present invention, wherein: the optimal reaction conditions are: the reaction temperature is 40 °C, the pH is 6.5, the substrate concentration of β-ionol is 2.5 mM, and NADP+ At a concentration of 1.0 mM, 0.29 mg / mL of NaSDR and 0.39 mg / mL of AaDBR1 were added, and the reaction time was 2 h.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides a method for biosynthesizing dihydro-β-ionone by a two-enzyme one-pot method, creating a new route and new technology for the preparation of dihydro-β-ionone. By searching for an alcohol dehydrogenase that can specifically catalyze the conversion of β-ionol to β-ionone and combining it with an artemisene acid reductase that has high activity in converting β-ionone to dihydro-β-ionone, an in vitro one-pot reaction was carried out, and a route and process for biologically preparing dihydro-β-ionone using β-ionol as a substrate were established, providing a method for realizing the green and sustainable large-scale synthesis of dihydro-β-ionone and its wide application as a natural product. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings. Among them:

[0019] Figure 1 Schematic diagram of the conversion of β-ionol to dihydro-β-ionone by a two-enzyme one-pot method;

[0020] Figure 2 Optimization diagram of the expression conditions of recombinant short-chain alcohol dehydrogenase NaSDR (a. Effect of induction time on the expression of NaSDR; b. Effect of induction temperature on the expression of NaSDR; c. Effect of IPTG concentration on the expression of NaSDR; d. Effect of induction timing on the expression of NaSDR);

[0021] Figure 3 SDS-PAGE diagram of recombinant short-chain alcohol dehydrogenase NaSDR (M: protein maker; 1: NaSDR whole cell; 2: NaSDR supernatant; 3: 100 mM imidazole; 4: 200 mM imidazole);

[0022] Figure 4 Optimization result diagram of the conditions for recombinant short-chain alcohol dehydrogenase NaSDR to catalyze β-ionol (a. Effect of temperature on the enzyme activity of NaSDR; b. Effect of pH on the enzyme activity of NaSDR; c. Temperature stability of NaSDR; d. pH stability of NaSDR; e. Effect of cofactor concentration on the enzyme activity of NaSDR; f. Effect of organic solvents on the enzyme activity of NaSDR);

[0023] Figure 5 It is the result diagram of cofactor dependence in the one-pot reaction system of NaSDR and AaDBR1;

[0024] Figure 6 It is the optimized result diagram of the reaction process for preparing dihydro-β-ionone by one-pot reaction of two enzymes;

[0025] Figure 7 It is the result diagram of the influence of different ADH enzymes on the yield of β-ionone (HPLC method). Specific Embodiments

[0026] To make the above objects, features and advantages of the present invention more obvious and understandable, the specific embodiments of the present invention will be described in detail below in conjunction with the embodiments of the specification.

[0027] Many specific details are set forth in the following description in order to fully understand the present invention, but the present invention can also be implemented in other ways different from those described herein. Those skilled in the art can make similar generalizations without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0028] Secondly, the so-called "one embodiment" or "embodiment" herein refers to a specific feature, structure or characteristic that can be included in at least one implementation manner of the present invention. The "in one embodiment" that appears in different places in this specification does not all refer to the same embodiment, nor is it an embodiment that is separate or selectively mutually exclusive with other embodiments.

[0029] Unless otherwise specified, the raw materials used in the examples are all commercially purchased.

[0030] In the examples, the culture media used are:

[0031] (1) Luria-Bertani (LB) medium: Weigh 10 g of Tryptone, 5 g of Yeast extract, 10 g of NaCl, dissolve in distilled water and make up to 1 L (adjust the pH to 7.0 - 7.5 with NaOH), and sterilize at 121 °C for 20 min. When preparing LB solid medium, add 20 g / L agar powder before autoclaving.

[0032] (2) SD medium: Weigh 1.7 g of YNB, 5 g of anhydrous ammonium sulfate, 20 g of agar powder, add 800 mL of water and sterilize at 115 °C for 15 min, then add 100 mL of 10× glucose stock solution and 100 mL of 10× amino acid stock solution.

[0033] Biological raw materials and their sources used in the embodiments of the present invention: β-ionol and β-ionone were both purchased from Sigma-Aldrich (USA).

[0034] In the present invention, a novel short-chain dehydrogenase NaSDR was cloned from the genome of Novosphingobium aromaticivorans DSM 12444, which has the function of specifically catalyzing the conversion of β-ionol to β-ionone. On this basis, a new route for the one-pot synthesis of dihydro-β-ionone from β-ionol was established by using the short-chain dehydrogenase NaSDR and the enoyl reductase AaDBR1 derived from Artemisia annua ( Figure 1 ). In the in vivo double-enzyme cascade reaction, the regeneration and self-sufficiency of NADPH were realized through the catalysis of NaSDR and AaDBR1, greatly saving the consumption of cofactors.

[0035] Example 1

[0036] (1) Construction of the expression vector of the short-chain alcohol dehydrogenase NaSDR

[0037] Using the NaSDR genome from Novosphingobium aromaticivorans DSM 12444 (the nucleotide sequence is shown in SEQ ID NO.3) as a template, the obtained NaSDR plasmid and pET28a were digested with NcoI and XholI double enzymes respectively. The recovered fragments were ligated with the pET-28a vector, and the product was transformed into Escherichia coli E.coli DH5α. The transformed product was spread on an LB plate and cultured overnight at 37°C. A single colony was inoculated into an LB (added kanamycin to a final concentration of 50 mg / L) liquid medium and cultured for 8-10 h. Then, the cells were collected to extract the plasmid, and the empty plasmid was verified and removed by enzyme digestion. The recombinant plasmid was subjected to nucleic acid sequence determination to obtain the correct recombinant expression vector pET-28a-NaSDR. The amino acid sequence of the short-chain alcohol dehydrogenase NaSDR from Novosphingobium aromaticivorans DSM12444 is shown in SEQ ID NO.1.

[0038] (2) Expression and purification of the recombinant short-chain alcohol dehydrogenase NaSDR

[0039] The recombinant Escherichia coli BL21(DE3) containing the plasmid pET-28a-NaSDR was streaked on an LB plate (the final concentration of kanamycin was 50 mg / L) for activation and cultured at 37°C for 12 h until a single colony grew. Then, it was inoculated into 50 mL of LB liquid medium and cultured at 37°C on a shaker at 180 rpm for 3-4 h until OD 600= 0.4 - 2.0. IPTG was added at a final concentration of 0.01 - 1.0 mM for induction, and the culture was continued at 28 °C for 16 h. The effects of induction temperature (16 - 37 °C) and induction time (8 - 28 h) on protein expression were investigated. The culture was centrifuged at 8000 rpm for 10 min to collect the bacterial cells. Meanwhile, Escherichia coli BL21(DE3) containing the empty plasmid pET28a was used as a negative control (Control). The optimized optimal culture conditions were: induction time of 12 h, induction temperature of 20 °C, final concentration of IPTG of 0.01 mM, OD 600 was 1.6, as Figure 2 . The culture was centrifuged at 8000 rpm for 10 min to collect the bacterial cells. Subsequently, the bacterial cells were resuspended in 1×PBS (pH 7.4) solution and sonicated at an appropriate power. The working conditions were: sonication for 3 s, interval of 3 s, for a total of 99 times. Centrifugation was carried out at 12000 r / min for 10 min, and the supernatant was aspirated to obtain the crude enzyme solution of recombinant NaSDR.

[0040] The obtained crude enzyme solution of recombinant NaSDR was passed through a 0.22 μm filter membrane and purified using a Ni-NTA pre-packed gravity column. First, 2 - 5 column volumes of 25 mM NaH2PO4-Na2HPO4 buffer were added to equilibrate the column, then 2 column volumes of the crude enzyme solution were added for binding. Finally, the recombinant protein was eluted with buffers containing different concentrations of imidazole (20, 50, 100, 200, and 400 mM), and the eluates with different concentrations of imidazole were collected separately. 15 μL of the crude enzyme solution of the transformant expressing the NaSDR gene and 15 μL of the purified recombinant NaSDR enzyme were each added with 15 μL of 5×SDS loading buffer, mixed well, boiled for 5 min, and then 10 μL of each was taken for SDS-PAGE electrophoresis to identify the expressed protein, as Figure 3 . It can be seen that an electrophoretically pure target protein, a target band of approximately 33 kDa, was obtained through affinity chromatography.

[0041] Example 2

[0042] (1) Enzyme activity assay method for recombinant NaSDR enzyme

[0043] The enzyme activity assay of recombinant NaSDR enzyme used β-ionol as the substrate. The 1 mL reaction system contained 2 mM β-ionol, 100 mM phosphate buffer (pH 7.0), 1 mM cofactor (NAD + or NADP + ) and 200 μL of pure enzyme. The centrifuge tube was placed at 35 °C for static reaction for 30 min, and then 1 mL of ethanol was added to terminate the reaction. An equal volume of distilled water was used to replace the pure enzyme solution as a blank control for the reaction.

[0044] The enzyme activity (U) of the recombinant NaSDR enzyme is defined as: the amount of enzyme required to oxidatively cleave β-ionol to produce 1 nmol of β-ionone per minute. The calculation of enzyme activity was carried out with reference to the β-ionone standard curve, and the standard curve was y = 25.414x - 51.193 (R 2 = 0.9999).

[0045] (2) Optimal reaction temperature of the recombinant NaSDR enzyme

[0046] In the range of 25 - 60 °C, at intervals of 5 °C, the enzyme activity was measured separately. The buffer was 100 mM citric acid - disodium hydrogen phosphate buffer (pH 7.0), and the highest enzyme activity obtained was taken as 100%. The experimental results are as Figure 4 shown, Figure 4 a shows the effect of temperature on the NaSDR enzyme activity. The results indicate that the optimal reaction temperature of the recombinant NaSDR enzyme is 35 °C.

[0047] (3) Optimal reaction pH of the recombinant NaSDR enzyme

[0048] Under the condition of the optimal temperature of 35 °C, the standard reaction system was placed in citric acid - sodium hydroxide buffer (pH 4.0 - 8.5), Tris - HCL buffer (pH 7.0 - 9.0), and glycine - sodium hydroxide buffer (pH 8.0 - 11.0) for reaction, and the highest enzyme activity obtained was taken as 100%. The experimental results are as Figure 4 shown, Figure 4 b shows the effect of pH on the NaSDR enzyme activity. The results indicate that the optimal reaction pH of the recombinant NaSDR enzyme is 6.5.

[0049] (4) Temperature stability of the recombinant NaSDR enzyme

[0050] In the absence of substrate and other cofactors, the enzyme was incubated in 100 mM phosphate buffer (pH 7.0) at 30, 35, and 40 °C for different times. The enzyme activity values at different temperatures and different incubation times were measured according to the standard method. Taking the enzyme activity without incubation as 100%, the relative enzyme activity was calculated. The experimental results are as Figure 4 shown, Figure 4 c shows the temperature stability of the NaSDR enzyme. The results indicate that after the purified NaSDR enzyme was incubated at 30 °C or 35 °C for 3 h, its residual activity remained above 70%. After incubation at 40 °C for 3 h, its remaining activity remained below 60%.

[0051] (5) pH stability of the recombinant NaSDR enzyme

[0052] In the absence of substrate and other cofactors, the residual activity of the enzyme was determined after incubation at 4 °C for 8 h in 100 mM phosphate buffer (pH 5.0 - 8.0) or 100 mM Tris-HCL buffer (pH 7.0 - 9.0) to determine the pH stability of NaSDR. Taking the enzyme activity of the unincubated enzyme as 100%, the relative enzyme activity was calculated. The experimental results are as Figure 4 shown, Figure 4 d is the pH stability of the NaSDR enzyme. The results show that the enzyme has good pH stability, and within the range of pH 5.5 - 8.0, its activity always remains above 80% of the initial activity.

[0053] (6) Effect of cofactor concentration on the enzyme activity of recombinant NaSDR enzyme

[0054] Different concentrations (0.125 - 2.000 mM) of NAD + and NADP + were added to the standard reaction system respectively, and taking the highest enzyme activity obtained as 100%, the relative enzyme activities at different concentrations were calculated. The experimental results are as Figure 4 shown, Figure 4 e is the effect of cofactor concentration on the NaSDR enzyme activity. The results show that NaSDR can use both NAD + and NADP + as cofactors, but its dependence on NAD + is much higher than that on NADP + .

[0055] (7) Effect of common organic solvents on the enzyme activity of recombinant NaSDR enzyme

[0056] 0 - 25% (v / v) ethanol, methanol, DMSO were added to the standard reaction system respectively, with a final concentration of 1 mM and 5 mM metal ions and the chemical reagent EDTA. The enzyme activity was measured according to the standard method. Taking the enzyme activity measured under the condition of not adding organic solvents, ions and chemical reagents as 100%, the relative enzyme activities under the condition of adding different concentrations of substances were calculated. The experimental results are as Figure 4 shown, Figure 4 f is the effect of organic solvents on the NaSDR enzyme activity. The results show that adding a small amount of DMSO and methanol does not affect the NaSDR activity, ethanol significantly inhibits the NaSDR activity, and when the ethanol concentration exceeds 20%, the NaSDR activity is almost completely lost.

[0057] Example 3

[0058] In vitro synthesis of dihydro-β-ionone by one-pot two-enzyme method

[0059] The total volume of the one-pot reaction system is 1 mL, which contains 100 mM phosphate buffer (pH 7.0), 2 mM β-ionol, 1 mM NADP + , 0.29 mg / mL of pure NaSDR enzyme and 0.39 mg / mL of pure AaDBR1 enzyme (the enoic acid reductase AaDBR1 from Artemisia annua, whose amino acid sequence is shown in SEQ ID NO.2 and nucleotide sequence is shown in SEQ ID NO.4). The Eppendorf tube was placed at 35 °C for reaction for 30 min, and then 1 mL of ethanol was added to terminate the reaction.

[0060] The yield of dihydro-β-ionone obtained was determined by the following method: using HPLC 1200 high performance liquid chromatography (Agilent, USA), XDB-C18 chromatographic column (4.6×250 mm; i.d., 5 μm), flow rate 1 mL / min, column temperature 30 °C. The HPLC detection of dihydro-β-ionone was carried out for a total time of 20 min. The mobile phase was water and acetonitrile with a ratio of 30%:70%, and the wavelengths were 200 nm respectively. The results showed that the yield of dihydro-β-ionone reached 0.26 mM.

[0061] Example 4

[0062] Based on Example 3, in this Example 4, the addition amounts of pure NaSDR enzyme and pure AaDBR1 enzyme were adjusted to find the optimal enzyme addition ratio for the reaction. As shown in Table 1, the addition amounts of pure NaSDR enzyme and pure AaDBR1 enzyme and the corresponding results of the yield of dihydro-β-ionone were recorded.

[0063] Table 1

[0064]

[0065]

[0066] As can be seen from Table 1, when 0.29 mg / mL of NaSDR and 0.39 mg / mL of AaDBR1 were added to the system, the yield of dihydro-β-ionone reached the highest.

[0067] Comparative Example 1

[0068] The initial total volume of the one-pot reaction system was 1 mL, which contained 100 mM phosphate buffer (pH 7.0), 2 mM β-ionol, 1 mM NADP + , 0.29 mg / mL of pure NaSDR enzyme. The Eppendorf tube was placed at 35 °C for reaction for 30 min, and then 1 mL of ethanol was added to terminate the reaction.

[0069] The yield of the obtained dihydro-β-ionone was determined, and the result showed that dihydro-β-ionone could not be detected.

[0070] Comparative Example 2

[0071] The initial total volume of the one-pot reaction system was 1 mL, which contained 100 mM phosphate buffer (pH 7.0), 2 mM β-ionol, 1 mM NADP + and 0.39 mg / mL of pure AaDBR1 enzyme. The Eppendorf tube was placed at 35 °C for reaction for 30 min, and then 1 mL of ethanol was added to terminate the reaction.

[0072] The yield of the obtained dihydro-β-ionone was determined, and the result showed that dihydro-β-ionone could not be detected.

[0073] Example 5

[0074] In this Example 5, on the basis of Example 3, the types of cofactors were adjusted.

[0075] The initial total volume of the one-pot reaction system was 1 mL, which contained 100 mM phosphate buffer (pH 7.0), 2 mM β-ionol, 1 mM cofactor (using NAD alone + , using NADP alone + , using NAD + in combination with NADPH (1:1), using NADP + in combination with NADPH (1:1), 0.29 mg / mL of pure NaSDR enzyme and 0.39 mg / mL of pure AaDBR1 enzyme. The Eppendorf tube was placed at 35 °C for reaction for 30 min, and then 1 mL of ethanol was added to terminate the reaction.

[0076] The concentrations of β-ionol, β-ionone, and dihydro-β-ionone were determined. The test method was as follows: For the detection of compounds, HPLC 1200 high-performance liquid chromatography (Agilent, USA) was used, with an XDB-C18 chromatographic column (4.6×250 mm; i.d., 5 μm), a flow rate of 1 mL / min, and a column temperature of 30 °C. The total time for HPLC detection of β-ionol was 20 min, the mobile phase was water and acetonitrile, with a ratio of 38%:62%, and the detection wavelength of β-ionol was 200 nm. The total time for HPLC detection of β-ionone and dihydro-β-ionone was 20 min, the mobile phase was water and acetonitrile, with a ratio of 30%:70%, and the detection wavelengths of dihydro-β-ionone and β-ionone were 200 nm and 300 nm, respectively.

[0077] The results are as Figure 5 shown. NaSDR for NAD +has a relatively high dependence. Adding 1 mM NAD to the same reaction conditions + in the reaction system, the efficiency of catalyzing the conversion of β-ionol to β-ionone is 1.5 times that of adding 1 mM NADP + in the catalytic efficiency. AaDBR1 only uses NADPH as a cofactor. In the absence of NADPH, the synthesis of dihydro-β-ionone is inhibited. Therefore, when only NAD + is added, no dihydro-β-ionone is produced in the reaction system. When only NADP + is added, about 0.2 mM dihydro-β-ionone can be formed in the reaction system, and the molar conversion rate is about 10%. Using NAD + in combination with NADPH (1:1), the molar conversion rate is about 14%. Using NADP + in combination with NADPH (1:1), the molar conversion rate is about 20%. Considering that the cofactors NADP + and NADPH can be recycled during the catalytic processes of NaSDR and AaDBR1 enzymes, the cofactor of the reaction system is finally selected as adding only NADP + .

[0078] Example 6

[0079] In this Example 6, on the basis of Example 3, the reaction conditions were optimized.

[0080] (1) Optimization of the pH of the reaction system

[0081] The total volume of the one-pot reaction system is 1 mL, which contains 100 mM phosphate buffer (pH 5.0 - 8.0), 2 mM β-ionol, 1 mM NADP + , 0.29 mg / mL of pure NaSDR enzyme and 0.39 mg / mL of pure AaDBR1 enzyme. The Eppendorf tube was placed in a reaction at 35 °C for 30 min, and then 1 mL of ethanol was added to terminate the reaction.

[0082] The experimental results are as Figure 6 shown,[[]] Figure 6 a is the optimization of the pH of the reaction system. The results show that the optimal pH for the one-pot reaction is 6.5.

[0083] (2) Optimization of the temperature of the reaction system

[0084] The total volume of the one-pot reaction system is 1 mL, which contains 100 mM phosphate buffer (pH 7.0), 2 mM β-ionol, 1 mM NADP +, 0.29 mg / mL of pure NaSDR enzyme and 0.39 mg / mL of pure AaDBR1 enzyme. Place the Eppendorf tube at 25 - 55 °C for reaction for 30 min, and then add 1 mL of ethanol to terminate the reaction.

[0085] The experimental results are as Figure 6 shown, Figure 6 b is the temperature optimization of the reaction system. The results show that the optimal temperature for the one-pot reaction is 40 °C.

[0086] (3) Concentration optimization of the cofactor NADP + in the reaction system

[0087] The total volume of the one-pot reaction system is 1 mL, which contains 100 mM phosphate buffer (pH 7.0), 2 mM β-ionol, 0.125 - 2.000 mM NADP + , 0.29 mg / mL of pure NaSDR enzyme and 0.39 mg / mL of pure AaDBR1 enzyme. Place the Eppendorf tube at 35 °C for reaction for 30 min, and then add 1 mL of ethanol to terminate the reaction.

[0088] The experimental results are as Figure 6 shown, Figure 6 c is the concentration optimization of the cofactor NADP + in the reaction system. The results show that the optimal NADP + concentration for the one-pot reaction is 1.0 mM.

[0089] (4) Substrate addition concentration optimization in the reaction system

[0090] The total volume of the one-pot reaction system is 1 mL, which contains 100 mM phosphate buffer (pH 7.0), 0.5 - 5.0 mM β-ionol, 1 mM NADP + , 0.29 mg / mL of pure NaSDR enzyme and 0.39 mg / mL of pure AaDBR1 enzyme. Place the Eppendorf tube at 35 °C for reaction for 30 min, and then add 1 mL of ethanol to terminate the reaction.

[0091] The experimental results are as Figure 6 shown, Figure 6 d is the substrate addition concentration optimization in the reaction system. The results show that the optimal substrate concentration for the one-pot reaction is 2.5 mM.

[0092] (5) Time optimization in the reaction system

[0093] The total volume of the one-pot reaction system is 1 mL, which contains 100 mM phosphate buffer (pH 7.0), 2 mM β-ionol, 1 mM NADP +, 0.29 mg / mL of pure NaSDR enzyme and 0.39 mg / mL of pure AaDBR1 enzyme. Place the Eppendorf tube at 35 °C and react for 0.5 - 4.0 h, then add 1 mL of ethanol to terminate the reaction.

[0094] The experimental results are as Figure 6 shown, Figure 6 e is the time optimization of the reaction system. The results show that the optimal time for the one-pot reaction is 2 h.

[0095] Therefore, the optimized best reaction system contains 0.29 mg / mL NaSDR, 0.39 mg / mL AaDBR1, 1 mM NADP + and 2.5 mM β-ionol. React at 40 °C. After 2 h, the reaction reaches the maximum molar conversion rate of 35.6%, and the yield of dihydro-β-ionone is 173.11 mg / L.

[0096] Comparative Example 3

[0097] Select 4 alcohol dehydrogenases from different sources, namely NaSDR from Novosphingobium aromaticivorans DSM 12444, SsADH from Sulfolobus solfataricus strain DSM 1617, SlscADH1 from Solanum lycopersicum, and RhADH from Rhodococcus ruber DSM 44541.

[0098] Study the effects of the four alcohol dehydrogenases on the in vitro catalysis of β-ionol. The specific method is to introduce the cloned ADHs from four different sources into Escherichia coli cells for whole-cell culture. After culturing to a certain condition, add the same concentration of β-ionol for shake-flask fermentation. The results are as Figure 7 shown. It can be seen that the NaSDR protein has the highest catalytic activity for β-ionol, while SlscADH1 and SsADH have no catalytic effect on β-ionol.

[0099] The present invention uses short-chain alcohol dehydrogenase and enoate reductase for cascade reaction, establishes a process for the one-pot in vitro biological preparation of dihydro-β-ionone with β-ionol as the substrate, and realizes the regeneration and self-circulation of NADPH. Thus, a new method for the efficient and low-cost biosynthesis of dihydro-β-ionone is created.

[0100] In the one-pot reaction established by the present invention, the self-circulation of NADPH is achieved through the catalysis of NaSDR and AaDBR1, which greatly reduces the consumption of NADPH and the preparation cost of dihydro-β-ionone. Finally, by optimizing the reaction conditions of the one-pot system (reaction temperature is 40 °C, pH is 6.5, β-ionol substrate concentration is 2.5 mM, NADP + concentration is 1.0 mM, adding 0.29 mg / mL NaSDR and 0.39 mg / mL AaDBR1, reaction time is 2 h), a process for the in vitro one-pot biological preparation of dihydro-β-ionone using β-ionol as the substrate is established, providing a new technology for the production of dihydro-β-ionone. Under suitable conditions, the highest yield of dihydro-β-ionone reaches 173.11 mg / L, and the molar conversion rate is 35.6%.

[0101] SEQ ID NO:1 NaSDR

[0102]

[0103]

[0104] SEQ ID NO:2 AaDBR1

[0105]

[0106] SEQ ID NO:3 NaSDR

[0107]

[0108] SEQ ID NO:4 AaDBR1

[0109]

[0110]

[0111] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not restrictive. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A method for preparing dihydro-β-ionone by biocatalytic conversion of β-ionol, characterized in that: include, β-ionol was used as substrate, short-chain alcohol dehydrogenase NaSDR and enoate reductase AaDBR1 were added to react at a temperature of 25-60°C, a pH of 4.0-11.0, and a reaction time of 0.5-3 h to obtain β-ionone. In the reaction system, the addition amount of short-chain alcohol dehydrogenase NaSDR was 0.18~0.35 mg / mL; the addition amount of enoate reductase AaDBR1 was 0.39~0.47 mg / mL; The reaction system also includes a cofactor, which is NADP + , the concentration of the cofactor is 1.0 mM; Wherein, the short-chain alcohol dehydrogenase NaSDR is derived from Novosphingobium aromaticivorans DSM12444, the amino acid sequence of which is shown in SEQ ID NO.1; The enoate reductase AaDBR1 is derived from Artemisia annua Artemisia annua , and its amino acid sequence is shown in SEQ ID NO.

2.

2. The method for preparing dihydro-β-ionone by biocatalytic conversion of β-ionol as claimed in claim 1, characterized in that: In the reaction system, the addition amount of short-chain alcohol dehydrogenase NaSDR was 0.29 mg / mL; the addition amount of enoate reductase AaDBR1 was 0.39 mg / mL.

3. The method for preparing dihydro-β-ionone by biocatalytic conversion of β-ionol as claimed in claim 1 or 2, characterized in that: The β-ionol substrate concentration is 0.5-5.0 mM.

4. The method for preparing dihydro-β-ionone by biocatalytic conversion of β-ionol as claimed in claim 3, characterized in that: The β-ionol substrate concentration was 2.5 mM.

5. The method for preparing dihydro-β-ionone by biocatalytic conversion of β-ionol as claimed in claim 1, characterized in that: The reaction temperature was 40°C, the pH was 6.5, and the reaction time was 2 h.

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