Alcohol dehydrogenase mutant with high nootkatone production and preparation method thereof

Abstract

The present invention discloses a high-yield nootkatone alcohol dehydrogenase mutant and a preparation method thereof, and is used to synthesize a high-yield nootkatone alcohol dehydrogenase mutant derived from Alpinia oxyphylla ( Alpinia oxyphylla ) alcohol dehydrogenase undergoes site-directed mutagenesis, wherein the mutation is at least one of S197V, M144C, and L196A. The alcohol dehydrogenase mutant provided by the present invention has high catalytic activity. The mutant M144C / L196A / S197V achieves a yield of 99% in the production of nootkatone from nootkadol, 4.32 times that of the original enzyme. The alcohol dehydrogenase mutant catalyzes the synthesis of nootkatone using a simple production process, mild reaction conditions, and an environmentally friendly production process, facilitating the industrial production of nootkatone and possessing broad application prospects.

Description

Technical Field

[0001] The invention relates to an alcohol dehydrogenase, in particular to an alcohol dehydrogenase that catalyzes nootkatone, and belongs to the technical fields of enzyme engineering and genetic engineering. Background Art

[0002] Nootkatone ((+)-Nootkatone), also known as citronellol, is found in plants such as grapefruit, Alaskan yellow cedar, vetiver, and alpinia. With its distinctive citrus aroma, Nootkatone is an important natural flavoring agent widely used in food, beverages, cosmetics, and perfumes. It also exhibits numerous biological activities, including insect repellent, antibacterial, and anti-inflammatory properties, making it valuable for applications in agriculture and medicine. However, the low concentration of nootkatone in plants makes direct isolation and extraction inefficient and costly, making it unsuitable for industrial applications.

[0003] There are many reports on the research of nootkatone synthesis technology, mainly based on chemical and biological synthesis methods. Chinese patent application CN 1830936A discloses a synthesis method of nootkatone and its application. It adopts a chemical synthesis method. Valerian oil is first extracted by supercritical technology to obtain valerian, and then a sodium hypochlorite / tert-butyl hydroperoxide mixed solution is added. After mixed reaction and separation and purification, nootkatone crystals are produced. Chinese patent application CN 108430464A uses β-pinene as raw material and utilizes ozone oxidation and decomposition to achieve high yield production of nootkatone. However, chemically synthesized nootkatone is a racemate with unstable purity and quality, which limits its application in high value-added products. In addition, the chemical synthesis method also has the disadvantages of complicated operation steps, harsh reaction conditions, and the need to use a large amount of organic solvents and catalysts, resulting in high production costs and serious environmental pollution.

[0004] Biosynthesis involves enzymes that catalyze the conversion of precursors such as limonene and nootkatol into nootkatone. Chinese patent application CN 115976118 A discloses a method and vector for the biosynthesis of nootkatone. Using genetic engineering techniques, the complete set of enzymes involved in nootkatone synthesis from Alpinia oxyphylla was introduced into expression cells, resulting in the construction of recombinant bacteria capable of synthesizing nootkatone from farnesyl pyrophosphate. Professor Liu Tiangang and his team significantly increased the yield of nootkatol synthesized from carbon sources to 10-15 g / L through carbon source selection and optimization, microbial strain modification, and fermentation condition optimization. They also synthesized nootkatone in yeast. However, under conventional reaction conditions, the low activity of alcohol dehydrogenase, which catalyzes the conversion of nootkatol to nootkatone, has become a major limitation in the biosynthetic production of nootkatone. Summary of the Invention

[0005] Purpose of the invention: The purpose of the present invention is to provide an alcohol dehydrogenase mutant that can produce high nootkatone production, and to provide a nucleic acid molecule encoding the mutant, a vector or recombinant cell or product comprising the mutant, and a preparation method and application of the mutant.

[0006] Technical solution: The first aspect of the present invention provides an alcohol dehydrogenase mutant with high nootkatone production, wherein the amino acid sequence of the alcohol dehydrogenase mutant is obtained by mutation of the sequence shown in SEQ ID NO.1, wherein the mutation is at least one of S197V, M144C, and L196A.

[0007] The wild-type alcohol dehydrogenase used in the present invention is derived from Alpinia oxyphylla and can catalyze the synthesis of nootkatone using nootkatol as a substrate and nicotinamide adenine dinucleotide oxidase (LpNOX) as a coenzyme. The present invention alters the protein structure and function by subjecting the wild-type alcohol dehydrogenase to site-directed mutagenesis of the amino acids in its active center. Screening revealed that single or combined mutations of S197V, M144C, or L196A can yield highly active alcohol dehydrogenase mutants. These mutants catalyze the production of nootkatone from nootkatol with significantly increased yields compared to the wild-type alcohol dehydrogenase.

[0008] The present invention uses the standard single-letter code for amino acids and the standard substitution notation, for example: S197V means that the serine (S) at position 197 at the N-terminus is mutated to valine (V); S197V / M144C means that the serine (S) at position 197 at the N-terminus is mutated to valine (V), and the methionine (M) at position 144 at the N-terminus is mutated to cysteine ​​(C).

[0009] In a second aspect, the present invention provides a nucleic acid molecule encoding the alcohol dehydrogenase mutant described in the first aspect. The nucleic acid molecule is capable of expressing the alcohol dehydrogenase mutant that produces high nootkatone production.

[0010] Furthermore, the nucleotide sequence of the nucleic acid molecule is obtained by base mutation of the sequence shown in SEQ ID NO.2.

[0011] In a third aspect, the present invention provides a vector comprising the nucleotide sequence described in the second aspect. The vector is a recombinant vector that can maintain its ability to replicate or autonomously replicate in various host cells, such as prokaryotic and / or eukaryotic cells, thereby amplifying or expressing the nucleotide sequence. The vector can be any conventional vector in the art, such as various plasmids, phages, or viral vectors.

[0012] Furthermore, the vector includes a cloning vector or an expression vector. The expression vector can be a PET series expression vector, preferably a pET22b(+) plasmid.

[0013] In a fourth aspect, the present invention provides a recombinant cell comprising the vector described in the third aspect. Preferably, the recombinant cell is Escherichia coli, such as Escherichia coli C43 or Escherichia coli BL21.

[0014] Furthermore, the method for constructing the recombinant cell comprises the following steps: (1) constructing an expression vector: connecting the nucleic acid molecule described in the second aspect with a plasmid to obtain the expression vector described in the third aspect; (2) constructing a recombinant cell: transferring the expression vector into competent cells, culturing and screening to obtain the recombinant cell.

[0015] In a fifth aspect, the present invention provides a method for preparing the alcohol dehydrogenase mutant according to the first aspect, comprising the following steps:

[0016] (1) Using the plasmid carrying the alcohol dehydrogenase gene as a template, a PCR reaction was performed using point mutation primers, and the mutant gene fragment and linearized plasmid were obtained after purification;

[0017] (2) Connecting the mutant gene fragment to the linearized plasmid to construct an expression vector, which is then transferred into the host bacteria for induced expression;

[0018] (3) Collecting the host bacteria expressing the alcohol dehydrogenase mutant, resuspending the bacteria and breaking the cells, and centrifuging and collecting the supernatant to obtain a crude enzyme solution containing the alcohol dehydrogenase mutant.

[0019] Furthermore, based on the preparation method, in step (3), the resuspension of the bacteria and subsequent operations can be omitted, and the collected wet bacteria can be directly used as whole-cell catalysts.

[0020] The crude enzyme solution or bacterial cell containing the alcohol dehydrogenase mutant obtained by the preparation method has high catalytic activity and can produce high yield of nootkatone.

[0021] Preferably, in step (1), the point mutation primers are as shown in Table 1:

[0022] Table 1 Point mutation primers

[0023] Primer name Sequence (5'-3') S197V_F gacccgcctgGTTatgccgcatc S197V_R gatgcggcatAACcaggcgggtc M144C_F ctatcgtgagcTGTgctagcgtgag M144C_R ctcacgctagcACAgctcacgatag L196A_F ccgacccgcGCTgttatgccgcatc L196A_R gatgcggcataacAGCgcgggtcgg

[0024] The uppercase letters in the primers indicate the mutation sites, “F” represents the upstream primer, and “R” represents the downstream primer.

[0025] Preferably, in step (1), the PCR reaction system is as shown in Table 2:

[0026] Table 2 PCR reaction system

[0027] Ingredients volume 10×BufferforKOD-Plus- 2.5 μL 2mM dNTP 2.5 μL <![CDATA[25mMMgSO4]]> 1.5 μL DMSO 1 μL 10 pmol / μL Forward Primer 0.75μL 10 pmol / μL Reverse Primer 0.75μL DNA template <100ng KOD-Plus- 1 μL <![CDATA[ddH2O]]> up to 25 μL

[0028] Preferably, in step (1), the conditions of the PCR reaction are as shown in Table 3:

[0029] Table 3 PCR reaction conditions

[0030]

[0031] In a sixth aspect, the present invention provides a product comprising the alcohol dehydrogenase mutant of the first aspect, or the nucleic acid molecule of the second aspect, or the vector of the third aspect, or the recombinant cell of the fourth aspect. The product includes a catalyst.

[0032] In a seventh aspect, the present invention provides a use of the product described in the sixth aspect in the catalytic preparation of nootkatone.

[0033] Furthermore, the catalytic substrate is nootkadol. The catalysis can be carried out in a potassium phosphate buffer solution. The catalytic reaction conditions include a temperature of 20-30°C and a pH of 7.0-9.0, and a reaction time of 8-16 hours. Preferably, the temperature is 25°C, the pH is 7.5, and the reaction time is 12 hours.

[0034] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The alcohol dehydrogenase mutant provided by the present invention has high catalytic activity towards nootkadol. The yield of nootkadone catalyzed by the mutant M144C / L196A / S197V can reach 99%, which is 4.32 times that of the original enzyme; 2. The production process of synthesizing nootkadone from nootkadol catalyzed by the alcohol dehydrogenase mutant provided by the present invention is simple, the reaction conditions are mild, and the production process is environmentally friendly, which is conducive to the industrial production of nootkadone and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a schematic diagram of the reaction of nootkatol to nootkatone catalyzed by alcohol dehydrogenase;

[0036] Figure 2 is the catalytic yield diagram of wild-type alcohol dehydrogenase and its mutants;

[0037] Figure 3 The catalytic yield of the alcohol dehydrogenase mutant M144C / L196A / S197V at different temperatures;

[0038] Figure 4 The catalytic yield of the alcohol dehydrogenase mutant M144C / L196A / S197V at different pH values ​​is shown;

[0039] Figure 5 The catalytic yield diagram of the alcohol dehydrogenase mutant M144C / L196A / S197V at different reaction times. DETAILED DESCRIPTION

[0040] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0041] The wild-type alcohol dehydrogenase and its mutants in the present invention can catalyze the synthesis of nootkatone ((+)-nootkatone) using nootkatol (trans-nootkatol) as a substrate and nicotinamide adenine dinucleotide oxidase (LpNOX) as a coenzyme. The reaction process is as follows: Figure 1 shown.

[0042] The materials used in the examples were obtained from:

[0043] 1. Strains and plasmids

[0044] Plasmids pET-22b(+), E. coli BL21(DE3), and E. coli DH5α were all deposited by the applicant and were commercially available. The wild-type alcohol dehydrogenase is AoSDR1ADH from Alpinia oxyphylla, and LpNOX is from Lactobacillus pentosus. The genes encoding both enzymes were synthesized by Genwi (Suzhou) Co., Ltd. Site-directed mutagenesis sequences for the enzymes were obtained by PCR and constructed by the applicant.

[0045] 2. Reagents and Culture Media

[0046] DNA gel recovery kit and plasmid extraction kit were both from Sangon Biotech (Shanghai) Co., Ltd.

[0047] The components of LB liquid culture medium are tryptone 10 g / L, yeast powder 5 g / L, and sodium chloride 10 g / L.

[0048] The components of LB solid medium are tryptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, and agar powder 15 g / L.

[0049] The components of TB liquid culture medium are yeast powder 12g / L, tryptone 12g / L, glycerol 4ml / L, dipotassium hydrogen phosphate 12.5g / L, and potassium dihydrogen phosphate 2.3g / L.

[0050] To obtain LpNOX powder: The pET-22b(+) plasmid containing the LpNOX gene was transformed into E. coli BL21(DE3) host cells and plated onto the surface of LB solid medium containing 100 μg / mL ampicillin. The cells were cultured at 37°C for 8 hours. A single E. coli colony was selected and inoculated into 3 mL of LB liquid medium containing 100 μg / mL ampicillin. The culture was incubated at 37°C overnight to serve as a seed solution. A 5% inoculum of the seed solution was transferred to a 250 mL Erlenmeyer flask containing 50 mL of TB medium and incubated at 37°C and 200 rpm. After 8 hours of culture, IPTG was added to a final concentration of 0.5 mM. The culture temperature was set to 18°C ​​and cultured for another 16 hours. The fermentation broth was centrifuged, the cells harvested, and resuspended in 200 mM phosphate buffer (pH 7.5). The resulting crude enzyme solution was ultrasonically disrupted and centrifuged. The supernatant was collected and lyophilized to obtain LpNOX powder.

[0051] Example 1 An alcohol dehydrogenase mutant S197V

[0052] In this example, alcohol dehydrogenase AoSDR1ADH was used as the original enzyme, and its amino acid sequence is shown in SEQ ID No. 1. The serine (S) at position 197 of its N-terminus was mutated to valine (V), resulting in the sequence of the mutant S197V. The nucleotide sequence of the original enzyme is shown in SEQ ID No. 2. The specific preparation method is as follows:

[0053] 1. Construction of recombinant plasmid

[0054] Using the pET-22b(+) plasmid carrying the alcohol dehydrogenase AoSDR1ADH gene as a template, PCR was performed using point mutation primers. The mutant gene fragment and linearized plasmid were purified. The point primers are shown in Table 4, the PCR reaction system is shown in Table 5, and the PCR reaction conditions are shown in Table 6.

[0055] Table 4 Primers for S197V mutant gene fragment and linearized plasmid

[0056] Primer name Sequence (5'-3') S197V_F gacccgcctgGTTatgccgcatc S197V_R gatgcggcatAACcaggcgggtc

[0057] Note: The uppercase letters in the primers indicate the mutation sites, “F” represents the upstream primer, and “R” represents the downstream primer.

[0058] Table 5 PCR reaction system

[0059] Ingredients volume 10×BufferforKOD-Plus- 2.5 μL 2mM dNTP 2.5 μL <![CDATA[25mMMgSO4]]> 1.5 μL DMSO 1 μL 10 pmol / μL Forward Primer 0.75μL 10 pmol / μL Reverse Primer 0.75μL DNA template <100ng KOD-Plus- 1 μL <![CDATA[ddH2O]]> up to 25 μL

[0060] Table 6 PCR reaction conditions

[0061]

[0062]

[0063] After verification by nucleic acid electrophoresis, the PCR product was purified using a DNA gel extraction kit. The reaction system shown in Table 7 was prepared, where the mutant gene fragment was a gene fragment in which serine at position 197 was mutated to valine, and the linearized plasmid was a linearized plasmid containing the gene fragment in which serine at position 197 was mutated to valine. The reaction was incubated at 37°C for 1 hour for one-step cloning to obtain a recombinant plasmid containing the mutant enzyme gene.

[0064] Table 7 One-step cloning system

[0065] Ingredients content Linearized plasmid 0.03 pmol Mutated gene fragment 0.06 pmol 5×CEIIBuffer 4 μL ExnaseII 2μL <![CDATA[ddH2O]]> to20μL

[0066] 2. Construction of recombinant strains and expression of enzyme mutants

[0067] Transform the recombinant plasmid into E. coli DH5α competent cells, spread the plasmid onto the surface of LB solid medium containing 100 μg / mL ampicillin, and incubate at 37°C for 12 hours. Scrape any single colonies growing on the surface of the plate into a test tube containing 5 mL of LB liquid medium containing 100 μg / mL ampicillin and incubate at 37°C with shaking at 200 rpm for 12 hours. Extract the plasmid using a plasmid extraction kit and store the recombinant plasmid at -20°C.

[0068] The recombinant plasmid was transformed into E. coli BL21(DE3) host cells and plated onto the surface of LB solid medium containing 100 μg / mL ampicillin and cultured at 37°C for 8 hours. A single E. coli colony was selected and inoculated into 3 mL of LB liquid medium containing 100 μg / mL ampicillin and cultured overnight at 37°C to serve as a seed culture. A 5% inoculum of the seed culture was transferred to a 250 mL Erlenmeyer flask containing 50 mL of TB medium and cultured at 37°C and 200 rpm. After 8 hours of culture, IPTG was added to a final concentration of 0.5 mM, the culture temperature was set to 18°C, and the culture was continued for 16 hours. The fermentation broth was centrifuged, the cells were collected, and resuspended in 200 mM phosphate buffer (pH 7.5) to obtain whole-cell catalyst (i.e., wet cells) containing the alcohol dehydrogenase mutant S197V.

[0069] Example 2 An alcohol dehydrogenase mutant S197V / M144C

[0070] In this example, the plasmid carrying the enzyme mutant S197V constructed in Example 1 was used as a template to mutate the methionine (M) at position 144 of its N-terminal amino acid sequence to cysteine ​​(C). The remaining preparation methods were the same as in Example 1. The primers for point mutations and plasmid construction are shown in Table 8.

[0071] Table 8 S197V / M144C mutant gene fragment primers and linearized plasmid primers

[0072]

[0073]

[0074] Example 3 An alcohol dehydrogenase mutant M144C / L196A / S197V

[0075] In this example, the plasmid carrying the enzyme mutant S197V / M144C constructed in Example 2 was used as a template to mutate the leucine (L) at position 196 of its N-terminal amino acid sequence to alanine (A). The remaining preparation methods were the same as in Example 2. The primers for point mutations and plasmid construction are shown in Table 9.

[0076] Table 9: Primers for M144C / L196A / S197V mutant gene fragments and linearized plasmid primers

[0077] Primer name Sequence (5'-3') L196A_F ccgacccgcGCTgttatgccgcatc L196A_R gatgcggcataacAGCgcgggtcgg

[0078] Example 4 Comparison of catalytic yields of wild-type alcohol dehydrogenase and its mutants

[0079] The wild-type alcohol dehydrogenase AoSDR1ADH and the whole-cell catalyst containing the alcohol dehydrogenase mutant prepared in Example 1-3 were respectively 600 =60, a substrate nootkatol concentration of 100 mM, a final LpNOX concentration of 5 mg / ml, a final NAD+ concentration of 80 μM, and a potassium phosphate buffer solution at pH 7.5. The reaction was carried out at 400 rpm overnight (approximately 12 hours) at room temperature (approximately 25°C). After completion of the reaction, a nootkatol-containing reaction solution was obtained. 500 μL of the nootkatol-containing reaction solution was extracted with 500 μL of ethyl acetate and centrifuged at 12,000 rpm for 1 minute. 300 μL of the supernatant was collected and analyzed by gas chromatography.

[0080] Gas chromatography analysis conditions: Agilent HP-5 column. GC program: injection pressure 23 psi, flow rate 2.5 ml / min, column temperature increased from 60°C at 50°C / min to 240°C, then held for 4 min. Single sample acquisition lasted 7.6 min. Nootkatone retention time was 5.24 min.

[0081] The test results are as follows Figure 2 The yields of the alcohol dehydrogenase mutants obtained in Examples 1-3 were all higher than those of the wild-type enzyme. The enzyme activity of the alcohol dehydrogenase mutant M144C / L196A / S197V obtained in Example 3 was significantly improved, and the nootkadone catalytic yield was 4.32 times that of the original enzyme, which is conducive to industrial production.

[0082] Example 5 Catalytic yield of alcohol dehydrogenase mutant M144C / L196A / S197V at different temperatures

[0083] Using the alcohol dehydrogenase mutant M144C / L196A / S197V from Example 3 as a catalyst, the same reaction system as in Example 4 was used. Reactions were carried out at 400 rpm overnight at 15°C, 20°C, 25°C, 30°C, 35°C, and 40°C. After completion of the reaction, a reaction solution containing nootkatone was obtained. The detection method was the same as in Example 4.

[0084] The test results are as follows Figure 3 Different temperatures have a significant effect on the catalytic activity of the alcohol dehydrogenase mutant enzyme M144C / L196A / S197V. The catalytic efficiency is better in the range of 15-30℃, and the enzyme activity decreases when the temperature is higher than 30℃.

[0085] Example 6 Catalytic Yield of Alcohol Dehydrogenase Mutant M144C / L196A / S197V at Different pH

[0086] The alcohol dehydrogenase mutant M144C / L196A / S197V of Example 3 was used as a catalyst. Based on the reaction system of Example 4, the catalyst OD 600 =40, the substrate nootkatol concentration was 80 mM, and potassium phosphate buffer solutions of pH = 6.0, 7.0, 8.0, 9.0, and 10.0 were reacted at 400 rpm at room temperature overnight to obtain a reaction solution containing nootkatol. The detection method was the same as in Example 4.

[0087] The test results are as follows Figure 4 As shown. The enzyme has good catalytic efficiency in the pH range of 8.0-9.0. In acidic buffer (pH = 6.0-7.0), the catalytic activity of the alcohol dehydrogenase mutant M144C / L196A / S197V is relatively limited. When the buffer pH is greater than 9.0, the enzyme activity gradually weakens.

[0088] Example 7 Catalytic yield of alcohol dehydrogenase mutant M144C / L196A / S197V at different reaction times

[0089] The alcohol dehydrogenase mutant M144C / L196A / S197V from Example 3 was used as a catalyst in the same reaction system as in Example 4. Reactions were performed at 400 rpm and room temperature for 1 h, 2 h, 4 h, 8 h, and 16 h, respectively. After completion of the reaction, a reaction solution containing nootkatone was obtained. The detection method was the same as in Example 4.

[0090] The test results are as follows Figure 5The optimal reaction time of the enzyme is 8-12 h. As the reaction time increases, the catalytic yield of nootkatone gradually increases and gradually levels off after 12 h.

[0091] Example 8 Catalytic Yield of Alcohol Dehydrogenase Mutant M144C / L196A / S197V at Different Cell Concentrations and Substrate Concentrations

[0092] The alcohol dehydrogenase mutant M144C / L196A / S197V of Example 3 was used as a catalyst. Based on the reaction system of Example 4, the catalyst OD 600 =20, 40, 60, and the substrate nootkadol concentration was 60mM, 80mM, and 100mM, and the reaction was carried out at 400 rpm at room temperature overnight to obtain a reaction solution containing nootkadol. The detection method was the same as that in Example 4.

[0093] The test results are shown in Table 10.

[0094] Table 10 The yield of nootkatone synthesized by alcohol dehydrogenase mutant M144C / L196A / S197V

[0095]

[0096] Example 9 Determination of Kinetic Parameters of Alcohol Dehydrogenase Mutant M144C / L196A / S197V

[0097] The specific activity of the alcohol dehydrogenase mutant M144C / L196A / S197V of Example 3 was measured at different substrate concentrations (2 mM, 5 mM, 10 mM, and 20 mM nocardol), and a double reciprocal curve was plotted based on the reciprocal of the specific activity and substrate concentration. The kinetic parameter Km was calculated to be 1.37 × 10 -15 mM, Kcat is 11.136S -1 .

Claims

1. A mutant of alcohol dehydrogenase capable of producing high-yield nootkatone, characterized in that: The amino acid sequence of the alcohol dehydrogenase mutant is obtained by mutating the sequence shown in SEQ ID NO.

1. The alcohol dehydrogenase mutant is S197V, S197V / M144C or M144C / L196A / S197V.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the alcohol dehydrogenase mutant according to claim 1.

3. A carrier, characterized in that Comprising the nucleic acid molecule of claim 2.

4. The carrier according to claim 3, characterized in that The vector includes a cloning vector or an expression vector.

5. A recombinant cell, characterized in that The recombinant cell comprises the vector according to claim 3.

6. A product, characterized in that The product comprises the alcohol dehydrogenase mutant according to claim 1 , the nucleic acid molecule according to claim 2 , the vector according to claim 3 , or the recombinant cell according to claim 5 .

7. Use of the product according to claim 6 in the catalytic preparation of nootkatone.

8. The use according to claim 7, characterized in that The catalytic substrate is nootkadol.

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