Use of isoeugenol monooxygenase mutants in isoeugenol vanillin production

Abstract

The application discloses application of isoeugenol monooxygenase mutants in isoeugenol vanillin production, and comprises recombinant strain construction, fermentation culture of the recombinant strain and catalytic reaction of a fermentation liquor, wherein the isoeugenol monooxygenase mutants are obtained by mutating isoeugenol monooxygenase amino acid sequences of wild-type bacteria WT and expressing the obtained bacterial bodies by using Escherichia coli, and a series of bacterial bodies which promote catalytic efficiency of vanillin production are formed. The application screens mutant super-high-efficiency enzyme catalysts with a catalytic conversion rate of 98.5% or above, avoids the shortcomings of traditional chemical catalyst production, such as low efficiency, pollution and high energy consumption, and makes the application more suitable for green industrial production requirements, so that the application is worthy of popularization and industrial use.

Description

Technical Field

[0001] This invention relates to the field of engineered bacteria and their industrial applications, and in particular to the application of isoeugenol monooxygenase mutants in the production of isoeugenol vanillin. Background Technology

[0002] Vanillin, a key natural flavoring agent, is widely used in food, beverages, cosmetics, and pharmaceuticals. Its traditional production method is primarily chemical synthesis, but with increasing emphasis on environmental protection and sustainable development, biosynthesis has gained widespread attention due to its clean and efficient characteristics. The production of vanillin via isoeugenol monooxygenase catalysis is one important biosynthetic pathway.

[0003] Isoeugenol, with the chemical formula C10H12O2, is a natural compound with a distinctive aroma. Its phenolic hydroxyl and methoxy groups make it readily oxidized by enzymes. Vanillin, scientifically known as 3-methoxy-4-hydroxybenzaldehyde, is one of the world's most produced and widely used fragrance compounds, possessing a rich vanilla bean aroma.

[0004] Monooxygenases are a class of enzymes that catalyze the introduction of an oxygen atom into a substrate molecule, and they participate in various metabolic processes in organisms. Isoeugenol monooxygenase is an enzyme that specifically catalyzes the oxidation of isoeugenol to vanillin. This enzyme is widely found in nature, such as in bacteria, fungi, and plants, but enzymes from different sources vary in catalytic efficiency, substrate specificity, and stability.

[0005] The oxidation of isoeugenol to vanillin by isoeugenol monooxygenase typically involves two steps: first, the enzyme catalyzes the oxidation of the phenolic hydroxyl group of isoeugenol to a quinone structure; second, the quinone structure is further hydrolyzed to generate vanillin. In this process, the enzyme binds to oxygen molecules and activates them into highly reactive oxygen free radicals, which then attack specific chemical bonds in the substrate molecule.

[0006] In recent years, scholars both domestically and internationally have made significant progress in the production of vanillin catalyzed by isoeugenol monooxygenase. For example, studies have reported the construction of yeast strains capable of efficiently expressing isoeugenol monooxygenase using genetic engineering techniques, achieving high yields of vanillin through optimized fermentation conditions. Under optimal reaction conditions, the conversion rate of isoeugenol can reach over 80%, and the molar yield of vanillin exceeds 70%.

[0007] The production of vanillin via isoeugenol monooxygenase catalysis is a green and efficient biosynthetic method with broad application prospects. Through continuous optimization of enzyme engineering, fermentation processes, and immobilization technology, it is expected to further improve the yield and purity of vanillin, reduce production costs, and meet market demand for high-quality vanillin. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the existing technology. By mutating the amino acid sequence of isoeugenol monooxygenase in wild fungus WT and expressing it in Escherichia coli, a series of bacterial cells that promote the catalytic efficiency of vanillin production are formed. In other words, the application of isoeugenol monooxygenase mutants in the production of isoeugenol vanillin is proposed.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] The isoeugenol monooxygenase mutant proposed in this invention is a mutant obtained by mutation and codon optimization based on the amino acid sequence shown in SEQ ID NO.1. The amino acid sequence shown in SEQ ID NO.1 is derived from the isoeugenol monooxygenase amino acid sequence of wild-type fungus WT. The obtained mutants include the following types:

[0011] WT as shown in SEQ ID NO.1 (expression record is strain 1);

[0012] For example, E47A as shown in SEQ ID NO.2: the glutamic acid E at position 47 of WT is mutated to alanine A (expression record is strain 2);

[0013] For example, T51A as shown in SEQ ID NO.3: The threonine T at position 51 of WT is mutated to alanine A (expression record is strain 3);

[0014] S122A, as shown in SEQ ID NO.4: The serine S at position 122 of WT is mutated to alanine A (expression record is strain 4);

[0015] For example, F216A as shown in SEQ ID NO.5: The phenylalanine F at position 216 of WT is mutated to alanine A (expression record is strain 5);

[0016] As shown in SEQ ID NO.6, W279A: the tryptophan W at position 279 of WT is mutated to alanine A (expression record is strain 6);

[0017] For example, F281A as shown in SEQ ID NO.7: the phenylalanine F at position 281 of WT is mutated to alanine A (expression record is strain 7);

[0018] For example, F355A as shown in SEQ ID NO.8: The phenylalanine F at position 355 of WT is mutated to alanine A (expression record is strain 8);

[0019] As shown in SEQ ID NO.9, C361A: the cysteine ​​C at position 361 of WT is mutated to alanine A (expression record is strain 9);

[0020] As shown in SEQ ID NO.10, C473A: the cysteine ​​C at position 473 of WT is mutated to alanine A (expression record is strain 10);

[0021] For example, T51A / F216A / W279A as shown in SEQ ID NO.11: the amino acids at positions 47, 51, 122, 216, 279, 281 and 355 of WT are all mutated to alanine A (expression record is strain 11);

[0022] As shown in SEQ ID NO.12, C361A / C473A: the amino acids at positions 361 and 473 of WT are both mutated to alanine A (expression record is strain 12);

[0023] As shown in SEQ ID NO.13, T51A / F216A / W279A / C361A / C473A: the amino acids at positions 47, 51, 122, 216, 279, 281, 355, 361 and 473 of WT are all mutated to alanine A (expression record is strain 13);

[0024] This invention also proposes the application of the aforementioned isoeugenol monooxygenase mutant in the production of isoeugenol vanillin, comprising the following steps:

[0025] S1. Construction of recombinant bacterial strains:

[0026] The isoeugenol monooxygenase mutant was expressed in Escherichia coli BL21(DE3) (preserved in our laboratory), and recombinant engineered strains were obtained by screening; glycerol bacteria were prepared from the recombinant engineered strains through culture.

[0027] S2. Fermentation culture of recombinant strains:

[0028] S201, Seed liquid preparation:

[0029] Take 6 glycerol bacteria, and inoculate 100 μl of each glycerol bacteria into 100 ml of M1 medium. Incubate at 35℃ and 220 rpm for 9 hours to obtain the resuscitation seed solution.

[0030] Subsequently, 1 ml of the revived seed solution was inoculated into 100 ml of M2 medium and cultured at 35°C and 220 rpm for 4 hours to prepare the expanded seed solution.

[0031] S202, Fermentation culture:

[0032] Prepare 2L M3 culture medium, weigh 20g of glucose and bring the volume to 25ml, then sterilize it separately and add it to the culture medium; set the initial fermentation conditions as follows: stirring speed 200rpm, aeration rate 2, tank pressure 0.25MPa, pH 7.0; maintain the dissolved oxygen at 30%, control the pH at 7.0 by adding ammonia water, and use 80% glucose solution for feeding;

[0033] S203, Cooling Induction:

[0034] When OD600 reaches 30, the fermenter temperature is lowered to 25°C, and IPTG with a final concentration of 0.6 mM / L is added to induce the expression of the target gene. The fermentation process lasts for about 24 hours until OD600 no longer increases. At this point, fermentation is stopped, and the fermentation broth is obtained. Subsequently, the fermentation broth is taken for protein gel electrophoresis analysis. The specific final OD values ​​and protein concentrations are detailed in Table 3.

[0035] S3, Catalytic reaction of fermentation broth:

[0036] The fermentation broth was adjusted to pH 10.5 with NaOH, and isoeugenol was added to a substrate concentration of 25 g / L for catalysis. The catalytic conditions were: 35℃, 180 rpm, maintaining pH 10.5, and continuous catalysis overnight for 16 h. Samples were taken after the reaction for HPLC analysis to determine the vanillin yield and conversion rate, as shown in Table 4.

[0037] Preferably, the preparation process of the recombinant engineered strain is as follows: the WT amino acid sequence was retrieved from the NCBI database (http: / / www.ncbi.nlm.nih.gov / ), with the number C3VA26.1; after mutation and codon optimization, the sequence was synthesized by Suzhou Genewise Biotechnology Co., Ltd., and the corresponding plasmid was constructed; as follows Figure 1 As shown in Table 2, these recombinant plasmids were transformed into Escherichia coli BL21(DE3) [purchased from Beijing Huayueyang Biotechnology, product code NRR01210, named BL21(DE3) Escherichia coli strain, and preserved in our laboratory] by heat shock method; through ampicillin resistance screening, we obtained recombinant engineered strains, and these recombinant bacteria are listed in Table 2; their correctness was verified by colony PCR method.

[0038] Preferably, the culturing process of the glycerol bacteria is as follows: the recombinant engineered bacteria are transferred from the plate to LB medium using an inoculation loop and cultured at 35°C and 220 rpm. When the OD600 reaches 1.0, glycerol is added and mixed well, then dispensed into cryovials and stored at -80°C for later use.

[0039] Furthermore, the LB medium comprises the following components at the following concentrations: 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride.

[0040] Preferably, the M1 culture medium comprises the following components at the following concentrations: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and the remainder being purified water;

[0041] Preferably, the M2 culture medium comprises the following components at the following concentrations: 16 g / L peptone, 10 g / L yeast extract, 10 g / L sodium chloride, and the remainder being purified water;

[0042] Preferably, the M3 culture medium comprises the following components at the following concentrations: 17 g / L disodium hydrogen phosphate dodecahydrate, 3 g / L dipotassium hydrogen phosphate, 0.5 g / L sodium chloride, 0.5 g / L magnesium sulfate heptahydrate, 10 g / L glucose, 15 g / L ammonium sulfate, 1 g / L ammonium chloride, with the remainder being purified water and ammonia water adjusting the pH to 7.0.

[0043] Preferably, based on catalytic yield and conversion rate, the isoeugenol monooxygenase mutants are specifically E47A, T51A, S122A, W279A, F355A, C361A, C473A, C361A / C473A, and T51A / F216A / W279A / C361A / C473A. The catalytic conversion rates of the above mutants are all >96%, among which C361A / C473A and T51A / F216A / W279A / C361A / C473A have the highest catalytic conversion rates (>98.5%). This is a highly efficient catalytic engineered bacteria project that is worthy of promotion to industrial use.

[0044] Compared with the prior art, the beneficial effects of the present invention are:

[0045] This invention improves the catalytic efficiency of the oxidation of isoeugenol to vanillin by mutating the amino acid sequence of isoeugenol monooxygenase and expressing it in Escherichia coli, making it more suitable for green industrial production. Through screening, a mutant ultra-efficient enzyme catalyst with a catalytic conversion rate of over 98.5% was obtained, avoiding the disadvantages of low efficiency, pollution and high energy consumption of traditional chemical catalysts, and is worthy of promotion to industrial use. Attached Figure Description

[0046] Figure 1 This is the plasmid transformation map obtained in Example 1 of the present invention;

[0047] Figure 2 This is a protein gel electrophoresis image of the fermentation broth in Example 2 of the present invention;

[0048] Figure 3This is an HPLC chromatogram showing the yield and conversion rate of vanillin in Example 3 of the present invention. Detailed Implementation

[0049] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with existing known technologies. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0050] The raw materials used in this invention (all prepared by mixing under aseptic conditions):

[0051] The LB medium formula is: 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride.

[0052] The formula for M1 medium is: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and the remainder is purified water;

[0053] The M2 medium formula is: 16 g / L peptone, 10 g / L yeast extract, 10 g / L sodium chloride, and the remainder is purified water;

[0054] The M3 culture medium formula is as follows: 17 g / L disodium hydrogen phosphate dodecahydrate, 3 g / L dipotassium hydrogen phosphate, 0.5 g / L sodium chloride, 0.5 g / L magnesium sulfate heptahydrate, 10 g / L glucose, 15 g / L ammonium sulfate, 1 g / L ammonium chloride, and the remainder is purified water. The pH is adjusted to 7.0 with ammonia.

[0055] The genes involved in this invention are shown in Table 1:

[0056] Table 1. Gene sequence of isoeugenol monooxygenase used in this invention

[0057] name amino acid sequence nucleotide sequence WT SEQ ID NO.1 SEQ ID NO.14 E47A SEQ ID NO.2 SEQ ID NO.15 T51A SEQ ID NO.3 SEQ ID NO.16 S122A SEQ ID NO.4 SEQ ID NO.17 F216A SEQ ID NO.5 SEQ ID NO.18 W279A SEQ ID NO.6 SEQ ID NO.19 F281A SEQ ID NO.7 SEQ ID NO.20 F355A SEQ ID NO.8 SEQ ID NO.21 C361A SEQ ID NO.9 SEQ ID NO.22 C473A SEQ ID NO.10 SEQ ID NO.23 E47A / T51A / S122A / F216A / w279A / F281A / F355A SEQ ID NO.11 SEQ ID NO.24 C361A / C473A SEQ ID NO.12 SEQ ID NO.25 E47A / T51A / S122A / F216A / w279A / F281A / F355A / C361A / C473A SEQ ID NO.13 SEQ ID NO.26

[0058] Example Group 1:

[0059] Construction of recombinant bacterial strains containing the isoeugenol monooxygenase gene sequence:

[0060] The amino acid sequence of the wild fungus WT required for this invention was retrieved from the NC BI database (website: http: / / www.ncbi.nlm.nih.gov / ), with the number C3VA26.1; after mutation and codon optimization, the sequence was synthesized by Suzhou Genewiz Biotechnology Co., Ltd., and the corresponding plasmid was constructed.

[0061] like Figure 1 As shown in Table 2, these recombinant plasmids were transformed into Escherichia coli BL21(DE3) [Product code: Beijing Huayueyang Biotechnology NRR01210, Product name: BL21(DE3) Escherichia coli strain, preserved in our laboratory] by heat shock method;

[0062] We obtained recombinant engineered strains through ampicillin resistance screening, and these recombinant strains are listed in Table 2. We also verified their correctness by colony PCR.

[0063] Thirteen strains were transferred from plates to LB medium using inoculation loops and cultured at 35°C and 220 rpm. When the OD600 reached 1.0, glycerol was added and mixed thoroughly. The mixture was then aliquoted into cryovials and stored at -80°C for later use.

[0064] Table 2. Names of Recombinant Bacterial Plasmids and Genes

[0065]

[0066] Example Group 2:

[0067] Fermentation culture of recombinant strains:

[0068] 1. Seed liquid preparation

[0069] Take 6 glycerol bacteria, inoculate 100 μl of each into 100 ml of M1 medium, and culture at 35 °C and 220 rpm for 9 hours to obtain the revived seed solution; then, take 1 ml of the revived seed solution and inoculate it into 100 ml of M2 medium, and continue to culture at 35 °C and 220 rpm for 4 hours to prepare the expansion seed solution.

[0070] 2. Fermentation culture

[0071] Prepare 2L M3 culture medium. Weigh 20g of glucose and bring the volume to 25ml, then sterilize it separately and add it to the medium. Initial fermentation conditions are set as follows: stirring speed 200rpm, aeration rate 2, tank pressure 0.25MPa, and pH 7.0. Maintain agitation-associated dissolved oxygen at 30%. Control the pH to 7.0 by adding ammonia and use 80% glucose solution for feeding.

[0072] 3. Cooling induction

[0073] When OD600 reached 30, the fermenter temperature was lowered to 25°C, and IPTG was added to a final concentration of 0.6 mM / L to induce target gene expression. Fermentation continued for approximately 24 hours until OD600 stopped increasing, at which point fermentation was stopped. Subsequently, the fermentation broth was analyzed by protein gel electrophoresis (refer to...). Figure 2 (Taking the fermentation broth of strain 13 as an example), the specific final OD value and protein concentration are detailed in Table 3.

[0074] Table 3. Final OD value and protein concentration of fermentation broth

[0075] strain <![CDATA[OD 600 ]]> Enzyme concentration (g / L) strain 1 140.53 6.13 strain 2 138.42 6.25 strain 3 143.45 6.41 strain 4 140.03 5.82 strain 5 138.31 5.73 strain 6 139.03 5.82 strain 7 143.56 5.93 strain 8 142.11 6.04 strain 9 139.26 5.83 strain 10 139.41 5.93 strain 11 140.32 6.24 strain 12 141.21 6.38 strain 13 143.53 6.50

[0076] Example Group 3:

[0077] Catalytic applications of recombinant bacterial fermentation broth:

[0078] The pH of each fermentation broth in Example Group 2 was adjusted to 10.5 using NaOH. Isoeugenol was added to a substrate concentration of 25 g / L for catalysis. The catalytic conditions were: 35°C, 180 rpm, maintaining pH 10.5, and continuous catalysis overnight for 16 h. Samples were taken after the reaction for HPLC analysis to determine the vanillin yield and conversion rate, as shown in Table 4.

[0079] Table 4. Yield and conversion rate of vanillin produced by catalytic fermentation broth

[0080] strain Yield (g / L) Conversion rate (%) strain 1 20.13 86.85％ strain 2 22.32 96.30％ strain 3 22.54 97.25％ strain 4 22.46 96.91％ strain 5 19.85 85.65％ strain 6 22.42 96.74％ strain 7 21.53 92.90％ strain 8 22.43 96.78％ strain 9 22.34 96.39％ strain 10 22.85 98.59％ strain 11 20.08 86.64％ strain 12 22.87 98.68％ strain 13 23.01 99.28％

[0081] As shown in Table 4, compared with the wild-type gene WT, the mutants C361A / C473A, which are transformed at amino acid positions 361 and 473, have a significant improvement in catalytic reaction, that is, a higher catalytic efficiency is achieved with less gene editing or modification, while T51A / F216A / W279A / C361A / C473A, which have completely changed characteristic amino acids, even reach a catalytic conversion rate of >99%.

[0082] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The application of isoeugenol monooxygenase mutant in the production of vanillin from isoeugenol, characterized in that, The amino acid sequence of the isoeugenol monooxygenase mutant is shown in SEQ ID NO.2 or SEQ ID NO.13; The application includes the following steps: S1. Construction of recombinant bacterial strains: The isoeugenol monooxygenase mutant was expressed in Escherichia coli BL21(DE3), and recombinant engineered strains were obtained through screening. Glycerol-containing bacteria were prepared from the recombinant engineered strains through culture. S2. Fermentation culture of recombinant strains: S201, Seed liquid preparation: Take 6 glycerol bacteria, and inoculate 100 μL of each glycerol bacteria into 100 mL of M1 medium. Incubate at 35 ℃ and 220 rpm for 9 hours to obtain the resuscitation seed solution. Subsequently, 1 mL of the revived seed solution was inoculated into 100 mL of M2 medium and cultured at 35 ℃ and 220 rpm for 4 hours to prepare the expanded seed solution; The M1 culture medium comprises the following components at the following concentrations: 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride, with the remainder being purified water; The M2 culture medium comprises the following components at the following concentrations: 16 g / L peptone, 10 g / L yeast extract, and 10 g / L sodium chloride, with the remainder being purified water; S202, Fermentation culture: Prepare 2L M3 culture medium, weigh 20 g of glucose and bring the volume to 25 mL, then sterilize it separately and add it to the culture medium; set the initial fermentation conditions as follows: stirring speed of 200 rpm, aeration rate of 2, tank pressure of 0.25 MPa, and pH value of 7.0; maintain the dissolved oxygen associated with stirring at 30%, control the pH value to 7.0 by adding ammonia water, and use 80% glucose solution for feeding; The M3 culture medium comprises the following components at the following concentrations: 17 g / L disodium hydrogen phosphate dodecahydrate, 3 g / L dipotassium hydrogen phosphate, 0.5 g / L sodium chloride, 0.5 g / L magnesium sulfate heptahydrate, 10 g / L glucose, 15 g / L ammonium sulfate, and 1 g / L ammonium chloride, with the remainder being purified water and ammonia water adjusted to pH 7.

0. S203, Cooling Induction: When OD600 reaches 30, the fermenter temperature is lowered to 25°C, and IPTG at a final concentration of 0.6 mM / L is added to induce the expression of the target gene. The fermentation process lasts for about 24 hours until OD600 no longer increases. At this point, the fermentation is stopped, and the fermentation broth is obtained. Subsequently, the fermentation broth is taken for protein gel electrophoresis analysis to obtain the final OD value and protein concentration. S3, Catalytic reaction of fermentation broth: Adjust the fermentation broth to pH 10.5 with NaOH, add isoeugenol to a substrate concentration of 25 g / L for catalysis. Catalysis conditions: 35℃, 180 rpm, maintain pH 10.5, catalyze continuously overnight for 16 h. After the reaction, take samples for HPLC detection of vanillin yield and conversion rate.

2. The application of the isoeugenol monooxygenase mutant according to claim 1 in the production of vanillin from isoeugenol, characterized in that, The preparation process of the recombinant engineered strain is as follows: after mutation and codon optimization, the nucleotide sequence shown in SEQ ID NO.15 or SEQ ID NO.26 is synthesized and the corresponding plasmid is constructed; the plasmid is transformed into Escherichia coli BL21(DE3) by heat shock method; the recombinant engineered strain is obtained by screening for ampicillin resistance; the correctness is verified by colony PCR method.

3. The application of the isoeugenol monooxygenase mutant according to claim 1 in the production of vanillin from isoeugenol, characterized in that, The culturing process of the glycerol bacteria is as follows: the recombinant engineered bacteria are transferred from the plate to LB medium using an inoculation loop and cultured at 35°C and 220 rpm; when the OD600 reaches 1.0, glycerol is added and mixed well, then dispensed into cryovials and stored at -80°C for later use.

4. The application of the isoeugenol monooxygenase mutant according to claim 3 in the production of vanillin from isoeugenol, characterized in that, The LB medium comprises the following components at the following concentrations: 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride.

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