Method for producing isoeugenol vanillin by using amycolatopsis
By constructing the recombinant phlomic acid-free strain for bioconversion of isoeugenol, the problems of high energy consumption, high pollution and high cost of traditional chemical synthesis of vanillin are solved, and the green biosynthesis and cost reduction of vanillin are achieved.
Patent Information
- Application Number
- CN202510094415.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-21
- Publication Date
- 2025-04-11
AI Technical Summary
Traditional methods of synthesis of vanillin have problems such as high energy consumption, high pollution and high cost.
Through gene knockout and recombinant technology, a recombinant plaque-free strain was constructed, and the strain was used for biotransformation of isoeugenol to produce vanillin.
The green biosynthesis of vanillin is achieved, reducing energy consumption and pollution, and has lower costs.
Smart Images
Figure CN119799807A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microbial fermentation, and more specifically to a method for producing isoeugenol vanillin by utilizing Amycolatopsis spp. Background Art
[0002] Vanillin is an organic compound that exudes a strong vanilla aroma. It is known as the "king of food flavors" and is widely used as a food flavoring agent around the world. It is not only widely used in food, daily chemicals, feed and other industries, but also plays an important role in the medical field. With the continuous growth of market demand for vanillin, biosynthetic methods for preparing vanillin are becoming more and more popular due to their environmental friendliness and sustainability.
[0003] Vanillin in the related art is prepared from phthalic anhydride, sodium hydroxide, chlorine, methanol and hydrochloric acid; phthalic anhydride, as a starting material, provides a basic carbon skeleton structure for the reaction and is the basic substance for synthesizing vanillin. The functional groups such as the benzene ring and anhydride group in its molecular structure will undergo a series of transformations in the subsequent reaction, gradually generating the molecular structure of vanillin; sodium hydroxide, as a strong base, can react with phthalic anhydride to play the role of ring opening and hydrolysis, thereby promoting the reaction to proceed in the direction of generating the corresponding intermediate products and creating conditions for the subsequent reaction; chlorine can react with certain intermediate products in the reaction system to undergo chlorination reaction Chlorine atoms are introduced to change the molecular structure and functional groups, making them closer to the structure of vanillin, laying the foundation for further reactions and functional group transformations; methanol, as a methylating agent, undergoes a methylation reaction with intermediate products containing functional groups such as hydroxyl groups, converting the corresponding functional groups into methoxy groups, making the structure and properties of the products closer to vanillin, and contributing to the final formation of vanillin; it can be used to adjust the pH of the reaction system so that the reaction proceeds under appropriate pH conditions, and it can also promote certain reaction steps, such as providing an acidic environment in reactions such as hydrolysis, prompting the corresponding chemical bonds to break and new functional groups to form.
[0004] However, there are still some disadvantages in its actual use, such as high energy consumption. The traditional chemical synthesis method of vanillin requires relatively harsh reaction conditions of high temperature and high pressure, and the reaction steps are complicated, resulting in high energy consumption; high pollution level. The traditional chemical synthesis method uses some toxic and harmful chemical reagents, such as chlorine used in the preparation of vanillin by phthalic anhydride. These substances are not only harmful to the environment, but may also remain in the product, posing a safety hazard; high cost. The raw materials of traditional chemical synthesis of vanillin, such as guaiacol, are mostly derived from petrochemical products, and the price is greatly affected by fluctuations in the oil market. As resources become increasingly scarce, the cost tends to rise. Summary of the invention
[0005] In order to improve the above problems and reduce the problems of high energy consumption, high pollution and high cost of vanillin in the related art, the present invention provides a method for producing isoeugenol vanillin using Amycolatopsis sp. to solve the problems raised in the above background technology.
[0006] To achieve the above object, the present invention provides the following technical solutions:
[0007] A method for producing isoeugenol vanillin by using Amycolatopsis spp. is prepared from a recombinant strain and an isoeugenol solution, and the preparation method comprises the following steps:
[0008] A1. Take out the AsPnIEM glycerol bacteria recombinant strain from the low temperature environment of -80°C, let it thaw naturally at room temperature, and after thawing, inoculate the strain on the surface of the activated solid culture medium, and culture it at 30°C for 3-4 days; use sterile tools to select a single colony from the plate, inoculate it into the seed culture medium, and culture it at a temperature of 30°C and a rotation speed of 220rpm for 24-48h until the OD600 value reaches 7, to obtain a seed solution;
[0009] A2, inoculating the seed liquid obtained in A1 into a 5L fermentation tank at an inoculation rate of 10%, and placing it in a 3L fermentation medium, adjusting the pH to 7.2, and then fermenting for 12-16h at a temperature of 30°C, a stirring speed of 500rpm, and a ventilation rate of 1-2vvm until the OD600 value reaches 3-4; at this time, adding a total amount of 90-150g of isoeugenol solution to the fermentation tank three times, and continuing the fermentation and conversion for 50h to obtain isoeugenol vanillin produced by using Amycolatopsis spp.
[0010] Preferably, the recombinant strain is prepared from a recombinant plasmid and Amycolatopsis spp.
[0011] Preferably, the raw material components and volume ratios of the recombinant strain are as follows: recombinant plasmid: Amycolatopsis is (1-3): (1-2).
[0012] Preferably, the method for preparing the recombinant strain comprises the following steps:
[0013] C1. Place 1 μL of vdhL-F primer, 1 μL of vdhL-R primer, 1 μL of vdh gene DNA template, 10 μL of DNA polymerase, and 7 μL of water in a PCR instrument, pre-denature at 90°C for 5 min, then denature at 95°C for 30 s, then anneal at 55°C for 30 s, repeat the above operation for 35 cycles, amplify the upstream sequence of the vdh gene at 72°C and a speed of 1 kb / min to obtain the upstream sequence of the vdh gene, and then use the same conditions to amplify the downstream sequence of the vdh gene using vdhR-F primer and vdhR-R primer to obtain the downstream sequence of the vdh gene.
[0014] C2. Mix 2 μL of pULcrRNA-km-Cas12 plasmid, 2 μL of PnIEM gene sequence, 2 μL of vdh gene upstream sequence, 2 μL of vdh gene downstream sequence, 2 μL of water and 10 μL of ligase and place in a PCR instrument. Perform seamless cloning reaction at 50°C for 30 minutes to obtain a recombinant plasmid named pULΔvdh:PnIEM.
[0015] C3, taking out the glycerol bacteria of Pseudomonas amycolata stored in a -80°C refrigerator and thawing it at room temperature; then, inoculating the strain onto a solid culture medium for activation culture, and continuing to culture at a temperature of 30°C for 3 days; then, inoculating the strain into an activation culture medium, and continuing to culture at a temperature of 30°C and a rotation speed of 220 rpm for 2 days; when the OD value reaches 0.7-0.8, collecting the bacteria to obtain Pseudomonas amycolata;
[0016] C4. After the recombinant plasmid pULΔvdh:PnIEM obtained in C2 is fully mixed with the Amycolatopsis pseudomycoticus obtained in C3, the mixture is electrotransformed under the conditions of an electric field strength of 20 kV / cm, a capacitance of 30 μF, a resistance of 300 Ω, and an electric shock time of 4 ms; then, the transformed cells are added to an activation medium and incubated at a temperature of 30°C and a rotation speed of 220 rpm for 24 hours. The incubated cells are spread on a solid culture medium containing 50 μg / ml apramycin and cultured at 30°C; after a single colony grows on the culture medium, a colony PCR test is performed using primers IEMcheck-F and IEMcheck-R to verify the correct insertion of the gene. After colony verification, the correct strain is confirmed to be added with glycerol and stored at -80°C for a long time to obtain a recombinant strain, which is named AsPnIEM.
[0017] Preferably, the formula of the solid culture medium is: 4 g / L glucose, 4 g / L yeast extract, 10 g / L malt extract powder, and 20 g / L agar powder.
[0018] Preferably, the formula of the activation culture medium is: 10 g / L glucose, 10 g / L glycerol, 1 g / L beef extract, 2 g / L peptone, and 1 g / L yeast extract.
[0019] Preferably, the fermentation medium formula is: sucrose 103 g / L, yeast extract 1.5 g / L, Na2HPO4 4 g / L, NaCl 0.2 g / L, KH2PO4 0.1 g / L, MgSO4·7H2O 0.2 g / L, CaCl2 0.05 g / L.
[0020] Preferably, the seed culture medium formula is: glucose 5g / L, yeast extract 10g / L, KH2PO41g / L, Na2HPO44g / L, MgSO4·7H2O 0.2g / L, NaCl 0.2g / L, CaCl20.05 g / L.
[0021] Technical effects and advantages of the present invention:
[0022] 1. The present invention achieves the goal of efficiently converting isoeugenol into vanillin by knocking out the gene at the position of the vanillin dehydrogenase gene and recombinantly introducing isoeugenol monooxygenase to construct a recombinant gene;
[0023] 2. The production process of the present invention utilizes Amycolatopsis to produce isoeugenol vanillin. The biotransformation process carried out in microbial cells has relatively mild fermentation conditions and does not require a large amount of additional energy input to maintain the reaction conditions, thereby reducing energy consumption. In addition, it is a biological fermentation process and does not require the use of such toxic and harmful reagents. The reaction conditions are mild, the process is green and environmentally friendly, and the pollution to the environment is small.
[0024] 3. The present invention adopts Amycolatopsis spp. with non-toxic substances and low vanillin degradation rate as a platform, and constructs a microbial synthesis chassis for high-yield isoeugenol vanillin by integrating isoeugenol monooxygenase into the genome of Amycolatopsis spp., thereby realizing the green biosynthesis industrial production of vanillin and reducing costs. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 This is the synthetic route of isoeugenol vanillin in the present invention.
[0026] Figure 2 This is the HPLC spectrum of vanillin detection in Example 1 of the present invention.
[0027] Figure 3 This is the plasmid map of pULΔvdh:PnIEM in the present invention. DETAILED DESCRIPTION
[0028] The present invention is further described in detail below in conjunction with the embodiments of the present invention. The raw materials used in the examples and embodiments of the present invention are all commercially available common materials unless otherwise specified below.
[0029] Preparation Example 1-5
[0030] A recombinant strain, the preparation components and their corresponding proportions are shown in the following table, and is prepared by the following preparation method:
[0031] C1. Place 1 μL of vdhL-F primer, 1 μL of vdhL-R primer, 1 μL of vdh gene DNA template, 10 μL of DNA polymerase, and 7 μL of water in a PCR instrument, pre-denature at 90°C for 5 min, then denature at 95°C for 30 s, then anneal at 55°C for 30 s, repeat the above operation for 35 cycles, amplify the upstream sequence of the vdh gene at 72°C and a speed of 1 kb / min to obtain the upstream sequence of the vdh gene, and then use the same conditions to amplify the downstream sequence of the vdh gene using vdhR-F primer and vdhR-R primer to obtain the downstream sequence of the vdh gene.
[0032] C2. Mix 2 μL of pULcrRNA-km-Cas12 plasmid, 2 μL of PnIEM gene sequence, 2 μL of vdh gene upstream sequence, 2 μL of vdh gene downstream sequence, 2 μL of water and 10 μL of ligase and place in a PCR instrument. Perform seamless cloning reaction at 50°C for 30 minutes to obtain a recombinant plasmid named pULΔvdh:PnIEM.
[0033] The plasmid map of pULΔvdh:PnIEM is as follows Figure 3 As shown;
[0034] C3. Take out the glycerol bacteria of Pseudomonas amycolata stored in a -80°C refrigerator and thaw it at room temperature; then, inoculate the strain onto a solid culture medium for activation culture, and continue to culture at a temperature of 30°C for 3 days; then, inoculate the strain into an activation culture medium, and continue to culture at a temperature of 30°C and a rotation speed of 220 rpm for 2 days; when the OD value reaches 0.7, collect the bacteria to obtain Pseudomonas amycolata.
[0035] The formula of the solid culture medium is: glucose 4g / L, yeast extract 4g / L, malt extract powder 10g / L, agar powder 20g / L;
[0036] The formula of the activation medium is: glucose 10g / L, glycerol 10g / L, beef extract 1g / L, peptone 2g / L, yeast extract 1g / L;
[0037] C4. After the recombinant plasmid pULΔvdh:PnIEM obtained in C2 is fully mixed with the Amycolatopsis pseudomycoticus obtained in C3, the mixture is electrotransformed under the conditions of an electric field strength of 20 kV / cm, a capacitance of 30 μF, a resistance of 300 Ω, and an electric shock time of 4 ms; then, the transformed cells are added to an activation medium and incubated at a temperature of 30°C and a rotation speed of 220 rpm for 24 hours. The incubated cells are spread on a solid culture medium containing 50 μg / ml apramycin and cultured at 30°C; after a single colony grows on the culture medium, a colony PCR test is performed using primers IEMcheck-F and IEMcheck-R to verify the correct insertion of the gene. After colony verification, the correct strain is confirmed to be added with glycerol and stored at -80°C for a long time to obtain a recombinant strain, which is named AsPnIEM.
[0038] Table: Components and volume ratios of raw materials in Preparation Examples 1-5
[0039]
[0040] Preparation Example 6
[0041] A recombinant strain, which is different from Preparation Example 1 in that the preparation method is as follows:
[0042] C1. Place 1 μL of vdhL-F primer, 1 μL of vdhL-R primer, 1 μL of vdh gene DNA template, 10 μL of DNA polymerase, and 7 μL of water in a PCR instrument, pre-denature at 90°C for 5 min, then denature at 95°C for 30 s, then anneal at 55°C for 30 s, repeat the above operation for 35 cycles, amplify the upstream sequence of the vdh gene at 72°C and a speed of 1 kb / min to obtain the upstream sequence of the vdh gene, and then use the same conditions to amplify the downstream sequence of the vdh gene using vdhR-F primer and vdhR-R primer to obtain the downstream sequence of the vdh gene.
[0043] C2. Mix 2 μL of pULcrRNA-km-Cas12 plasmid, 2 μL of PnIEM gene sequence, 2 μL of vdh gene upstream sequence, 2 μL of vdh gene downstream sequence, 2 μL of water and 10 μL of ligase and place in a PCR instrument. Perform seamless cloning reaction at 50°C for 30 minutes to obtain a recombinant plasmid named pULΔvdh:PnIEM.
[0044] C3. Take out the glycerol bacteria of Pseudomonas amycolata stored in a -80°C refrigerator and thaw it at room temperature; then, inoculate the strain onto a solid culture medium for activation culture, and continue to culture at a temperature of 30°C for 3 days; then, inoculate the strain into an activation culture medium, and continue to culture at a temperature of 30°C and a rotation speed of 220 rpm for 2 days; when the OD value reaches 0.75, collect the bacteria to obtain Pseudomonas amycolata.
[0045] C4. After the recombinant plasmid pULΔvdh:PnIEM obtained in C2 is fully mixed with the Amycolatopsis pseudomycoticus obtained in C3, the mixture is electrotransformed under the conditions of an electric field strength of 20 kV / cm, a capacitance of 30 μF, a resistance of 300 Ω, and an electric shock time of 4 ms; then, the transformed cells are added to an activation medium, incubated at a temperature of 30°C and a rotation speed of 220 rpm for 24 hours, and the incubated cells are spread on a solid culture medium containing 50 μg / ml apramycin and cultured at 30°C; after a single colony grows on the culture medium, a colony PCR test is performed using primers IEMcheck-F and IEMcheck-R in the same manner as C1 to verify the correct insertion of the gene. After colony verification, the correct strain is confirmed to be added with glycerol and stored at -80°C for a long time to obtain a recombinant strain, which is named AsPnIEM.
[0046] Preparation Example 7
[0047] A recombinant strain, which is different from Preparation Example 1 in that the preparation method is as follows:
[0048] C1. Place 1 μL of vdhL-F primer, 1 μL of vdhL-R primer, 1 μL of vdh gene DNA template, 10 μL of DNA polymerase, and 7 μL of water in a PCR instrument, pre-denature at 90°C for 5 min, then denature at 95°C for 30 s, then anneal at 55°C for 30 s, repeat the above operation for 35 cycles, amplify the upstream sequence of the vdh gene at 72°C and a speed of 1 kb / min to obtain the upstream sequence of the vdh gene, and then use the same conditions to amplify the downstream sequence of the vdh gene using vdhR-F primer and vdhR-R primer to obtain the downstream sequence of the vdh gene.
[0049] C2. Mix 2 μL of pULcrRNA-km-Cas12 plasmid, 2 μL of PnIEM gene sequence, 2 μL of vdh gene upstream sequence, 2 μL of vdh gene downstream sequence, 2 μL of water and 10 μL of ligase and place in a PCR instrument. Perform seamless cloning reaction at 50°C for 30 minutes to obtain a recombinant plasmid named pULΔvdh:PnIEM.
[0050] C3. Take out the glycerol bacteria of Pseudomonas amycolata stored in a -80°C refrigerator and thaw it at room temperature; then, inoculate the strain onto a solid culture medium for activation culture, and continue to culture at a temperature of 30°C for 3 days; then, inoculate the strain into an activation culture medium, and continue to culture at a temperature of 30°C and a rotation speed of 220 rpm for 2 days; when the OD value reaches 0.8, collect the bacteria to obtain Pseudomonas amycolata.
[0051] C4. After the recombinant plasmid pULΔvdh:PnIEM obtained in C2 is fully mixed with the Amycolatopsis pseudomycoticus obtained in C3, the mixture is electrotransformed under the conditions of an electric field strength of 20 kV / cm, a capacitance of 30 μF, a resistance of 300 Ω, and an electric shock time of 4 ms; then, the transformed cells are added to an activation medium and incubated at a temperature of 30°C and a rotation speed of 220 rpm for 24 hours. The incubated cells are spread on a solid culture medium containing 50 μg / ml apramycin and cultured at 30°C; after a single colony grows on the culture medium, a colony PCR test is performed using primers IEMcheck-F and IEMcheck-R to verify the correct insertion of the gene. After colony verification, the correct strain is confirmed to be added with glycerol and stored at -80°C for a long time to obtain a recombinant strain, which is named AsPnIEM.
[0052] Preparation Example 8
[0053] A method for producing isoeugenol vanillin by using Amycolatopsis spp. is prepared by the following preparation method:
[0054] A1. Take out the AsPnIEM glycerol bacteria recombinant strain from the low-temperature environment of -80℃, let it thaw naturally at room temperature, and after thawing, inoculate the strain on the surface of the activated solid culture medium and culture it at 30℃ for 3 days; use sterile tools to pick a single colony from the plate, inoculate it into the seed culture medium, and culture it at 30℃ and 220rpm for 24h until the OD 600 When the value reaches 7, seed solution is obtained;
[0055] A2, inoculate the seed solution obtained in A1 into a 5L fermenter at a 10% inoculation rate, and place it in a 3L fermentation medium, adjust the pH to 7.2, and then ferment for 12h at a temperature of 30°C, a stirring speed of 500rpm, and a ventilation rate of 1vvm until the OD 600 The value reaches 3; at this time, a total of 90 g of isoeugenol solution was added to the fermentation tank three times, and the fermentation conversion was continued for 50 hours to obtain the production of isoeugenol vanillin by using Amycolatopsis spp.
[0056] Wherein AsPnIEM glycerol bacteria are prepared by Preparation Example 1;
[0057] The activated solid culture medium formula is: glucose 10g / L, glycerol 10g / L, beef extract 1g / L, peptone 2g / L, yeast extract 1g / L, agar powder 20g / L;
[0058] The seed culture medium formula is: glucose 5g / L, yeast extract 10g / L, KH2PO41g / L, Na2HPO44g / L, MgSO4·7H2O 0.2g / L, NaCl 0.2g / L, CaCl20.05 g / L;
[0059] The fermentation medium formula is: sucrose 103g / L, yeast extract 1.5g / L, Na2HPO44 g / L, NaCl 0.2g / L, KH2PO40.1 g / L, MgSO4·7H2O 0.2g / L, CaCl20.05 g / L;
[0060] The synthetic pathway of isoeugenol vanillin is as follows Figure 1 As shown;
[0061] Preparation Example 9
[0062] A method for producing isoeugenol vanillin using Amycolatopsis spp. is different from Preparation Example 8 in that the preparation method is as follows:
[0063] A1. Take out the AsPnIEM glycerol bacteria recombinant strain from the low-temperature environment of -80℃, let it thaw naturally at room temperature, and after thawing, inoculate the strain on the surface of the activated solid culture medium and culture it at 30℃ for 4 days; use sterile tools to pick a single colony from the plate, inoculate it into the seed culture medium, and culture it at 30℃ and 220rpm for 24h until the OD 600 When the value reaches 7, seed solution is obtained;
[0064] A2, inoculate the seed solution obtained in A1 into a 5L fermenter at a 10% inoculation rate, and place it in a 3L fermentation medium, adjust the pH to 7.2, and then ferment for 12h at a temperature of 30°C, a stirring speed of 500rpm, and a ventilation rate of 1vvm until the OD 600 The value reaches 3; at this time, a total of 90 g of isoeugenol solution was added to the fermentation tank three times, and the fermentation conversion was continued for 50 hours to obtain the production of isoeugenol vanillin by using Amycolatopsis spp.
[0065] Preparation Example 10
[0066] A method for producing isoeugenol vanillin using Amycolatopsis spp. is different from Preparation Example 8 in that the preparation method is as follows:
[0067] A1. Take out the AsPnIEM glycerol bacteria recombinant strain from the low-temperature environment of -80℃, let it thaw naturally at room temperature, and after thawing, inoculate the strain on the surface of the activated solid culture medium and culture it at 30℃ for 3 days; use sterile tools to pick a single colony from the plate, inoculate it into the seed culture medium, and culture it at 30℃ and 220rpm for 48h until the OD 600 When the value reaches 7, seed solution is obtained;
[0068] A2, inoculate the seed solution obtained in A1 into a 5L fermenter at a 10% inoculation rate, and place it in a 3L fermentation medium, adjust the pH to 7.2, and then ferment for 12h at a temperature of 30°C, a stirring speed of 500rpm, and a ventilation rate of 1vvm until the OD 600 The value reaches 3; at this time, a total of 90 g of isoeugenol solution was added to the fermentation tank three times, and the fermentation conversion was continued for 50 hours to obtain the production of isoeugenol vanillin by using Amycolatopsis spp.
[0069] Preparation Example 11
[0070] A method for producing isoeugenol vanillin using Amycolatopsis spp. is different from Preparation Example 8 in that the preparation method is as follows:
[0071] A1. Take out the AsPnIEM glycerol bacteria recombinant strain from the low-temperature environment of -80℃, let it thaw naturally at room temperature, and after thawing, inoculate the strain on the surface of the activated solid culture medium and culture it at 30℃ for 3 days; use sterile tools to pick a single colony from the plate, inoculate it into the seed culture medium, and culture it at 30℃ and 220rpm for 24h until the OD 600 When the value reaches 7, seed solution is obtained;
[0072] A2: The seed solution obtained in A1 was inoculated into a 5L fermenter at a 10% inoculation rate and placed in a 3L fermentation medium. The pH was adjusted to 7.2, and then fermented for 14 h at a temperature of 30°C, a stirring speed of 500 rpm, and a ventilation rate of 1.5 vvm until the OD 600 The value reached 3.5; at this time, a total of 90 g of isoeugenol solution was added to the fermentation tank three times, and the fermentation conversion was continued for 50 hours to obtain the production of isoeugenol vanillin using Amycolatopsis spp.
[0073] Preparation Example 12
[0074] A method for producing isoeugenol vanillin using Amycolatopsis spp. is different from Preparation Example 8 in that the preparation method is as follows:
[0075] A1. Take out the AsPnIEM glycerol bacteria recombinant strain from the low-temperature environment of -80℃, let it thaw naturally at room temperature, and after thawing, inoculate the strain on the surface of the activated solid culture medium and culture it at 30℃ for 3 days; use sterile tools to pick a single colony from the plate, inoculate it into the seed culture medium, and culture it at 30℃ and 220rpm for 24h until the OD 600 When the value reaches 7, seed solution is obtained;
[0076] A2: The seed solution obtained in A1 was inoculated into a 5L fermenter at a 10% inoculation rate and placed in a 3L fermentation medium. The pH was adjusted to 7.2, and then fermented for 16 h at a temperature of 30°C, a stirring speed of 500 rpm, and a ventilation rate of 2 vvm until the OD 600 The value reaches 4; at this time, a total of 90 g of isoeugenol solution was added to the fermentation tank three times, and the fermentation conversion was continued for 50 hours to obtain the production of isoeugenol vanillin using Amycolatopsis spp.
[0077] Preparation Example 13
[0078] A method for producing isoeugenol vanillin using Amycolatopsis spp. is different from Preparation Example 8 in that the preparation method is as follows:
[0079] A1. Take out the AsPnIEM glycerol bacteria recombinant strain from the low-temperature environment of -80℃, let it thaw naturally at room temperature, and after thawing, inoculate the strain on the surface of the activated solid culture medium and culture it at 30℃ for 3 days; use sterile tools to pick a single colony from the plate, inoculate it into the seed culture medium, and culture it at 30℃ and 220rpm for 24h until the OD 600 When the value reaches 7, seed solution is obtained;
[0080] A2, inoculate the seed solution obtained in A1 into a 5L fermenter at a 10% inoculation rate, and place it in a 3L fermentation medium, adjust the pH to 7.2, and then ferment for 12h at a temperature of 30°C, a stirring speed of 500rpm, and a ventilation rate of 1vvm until the OD 600 The value reaches 3; at this time, a total of 120 g of isoeugenol solution was added to the fermentation tank three times, and the fermentation conversion was continued for 50 hours to obtain the production of isoeugenol vanillin using Amycolatopsis sp.
[0081] Preparation Example 14
[0082] A method for producing isoeugenol vanillin using Amycolatopsis spp. is different from Preparation Example 8 in that the preparation method is as follows:
[0083] A1. Take out the AsPnIEM glycerol bacteria recombinant strain from the low-temperature environment of -80℃, let it thaw naturally at room temperature, and after thawing, inoculate the strain on the surface of the activated solid culture medium and culture it at 30℃ for 3 days; use sterile tools to pick a single colony from the plate, inoculate it into the seed culture medium, and culture it at 30℃ and 220rpm for 24h until the OD 600 When the value reaches 7, seed solution is obtained;
[0084] A2, inoculate the seed solution obtained in A1 into a 5L fermenter at a 10% inoculation rate, and place it in a 3L fermentation medium, adjust the pH to 7.2, and then ferment for 12h at a temperature of 30°C, a stirring speed of 500rpm, and a ventilation rate of 1vvm until the OD 600 The value reaches 3; at this time, a total of 150 g of isoeugenol solution was added to the fermentation tank three times, and the fermentation conversion was continued for 50 hours to obtain the production of isoeugenol vanillin using Amycolatopsis sp.
[0085] Preparation Examples 15-20
[0086] A method for producing isoeugenol vanillin using Amycolatopsis spp. is different from Preparation Example 8 in that the use of the recombinant strains used in its components is different, and the specific corresponding relationships are shown in the following table.
[0087] Table: Comparison of the use of recombinant strains in preparation examples 15-20
[0088] Group Recombinant strains Preparation Example 15 Prepared from Preparation Example 2 Preparation Example 16 Prepared from Preparation Example 3 Preparation Example 17 Prepared by Preparation Example 4 Preparation Example 18 Prepared from Preparation Example 5 Preparation Example 19 Prepared from Preparation Example 6 Preparation Example 20 Prepared from Preparation Example 7
[0089] Performance testing
[0090] The isoeugenol vanillin produced by using Amycolatopsis prepared in each embodiment was selected for testing, and the test objects were 130 portions of isoeugenol vanillin produced by using Amycolatopsis, 10 portions in each group; the yield, concentration and molar conversion rate were tested, and the specific testing steps were as follows:
[0091] First, the isoeugenol vanillin produced by using Amycolatopsis spp. prepared in the embodiment was sampled, 1 mL of fermentation broth was taken, centrifuged at 12000 rpm for 5 min, diluted 10 times and filtered using a 0.22 μM water filter membrane, and analyzed by HPLC; using a C18 column, 10 μL was injected, the column temperature was set to 35 ° C, the mobile phase was 30% methanol + 0.1% acetic acid, the flow rate was 1 mL / min, and the detection wavelength was 280 nm. The analysis was performed to calculate its yield, concentration and molar conversion rate.
[0092] It should be specifically noted that the isoeugenol vanillin produced by Pseudomonas amycolata is produced by Pseudomonas amycolata in a normal production mode, and the defective isoeugenol vanillin produced by Pseudomonas amycolata is discarded.
[0093] Examples 1-7
[0094] The corresponding relationship of the preparation method of producing isoeugenol vanillin by using Amycolatopsis spp. is shown in the following table.
[0095] Table: Comparison table of the use of isoeugenol vanillin produced by using Amycolatopsis in Examples 1-7
[0096] Group Production of isoeugenol vanillin by using Amycolatopsis spp. Example 1 Prepared from Preparation Example 8 Example 2 Prepared from Preparation Example 9 Example 3 Prepared from Preparation Example 10 Example 4 Prepared from Preparation Example 11 Example 5 Prepared from Preparation Example 12 Example 6 Prepared from Preparation Example 13 Example 7 Prepared from Preparation Example 14
[0097] The isoeugenol vanillin produced by Amycolatopsis sp. in Examples 1-7 above was selected, and its yield, concentration and molar conversion rate were tested according to the above measurement steps. The average values of the test results were recorded in the following table.
[0098] Table: Performance test results of yield, concentration and molar conversion of Examples 1-7
[0099]
[0100]
[0101] As can be seen from the above table, in the preparation process of isoeugenol vanillin produced by using Amycolatopsis in Examples 1-7, the effect of improving the production effect of isoeugenol vanillin produced by using Amycolatopsis is good, by using Amycolatopsis with non-toxic substances and low vanillin degradation rate as a platform. By integrating isoeugenol monooxygenase into the genome of Amycolatopsis strain, a microbial synthesis chassis of high-yield isoeugenol vanillin is constructed, thereby achieving the purpose of improving the production effect of isoeugenol vanillin produced by using Amycolatopsis;
[0102] Its yield is 81.14-134.82g; its concentration is 27.05-44.94mm / s; its molar conversion rate is 96.94-98.18%;
[0103] It can be seen that when the production raw materials are certain, the production effect of isoeugenol vanillin produced by using Pseudomonas amycolata can be increased by adjusting the preparation conditions. Combining the data in the above table, it is not difficult to see that when using Pseudomonas amycolata to produce isoeugenol vanillin, increasing the activation process culture time, seed liquid culture process culture time, fermentation process ventilation rate, fermentation time, fermentation result OD 600The value can increase the yield, concentration and molar conversion rate of vanillin. The reason is that extending the activation culture time can give Pseudomonas amycolata more time to adapt to the new environment and completely recover from the dormant state; increasing the seed liquid culture time can make the bacteria grow more fully and achieve a better growth state; increasing the ventilation rate can provide more sufficient oxygen for Pseudomonas amycolata in the fermentation system; extending the fermentation time means giving Pseudomonas amycolata more time to convert the substrate; higher OD 600 The values indicate that there are more bacteria in the fermentation system, which greatly improves the flux of substrate conversion, as obtained from Examples 1-5.
[0104] It can be seen that when the production raw materials are certain, the production effect of isoeugenol vanillin produced by Pseudomonas amycolata can be increased by adjusting the preparation conditions. It is not difficult to see from the data in the above table that when Pseudomonas amycolata is used to produce isoeugenol vanillin, when the isoeugenol input amount is 120g, the molar conversion rate is the highest. The reason for this is that compared with the input amount of 90g, 120g of isoeugenol provides more abundant substrates for Pseudomonas amycolata, which meets the needs of the thalli for further exerting the conversion ability; on the basis that the thalli cultured in the early stage have a certain conversion potential, more substrates increase the raw materials that can be converted into vanillin, and according to the chemical reaction process, there are more opportunities to generate products, which is beneficial to improve the molar conversion rate; at the same time, unlike the 150g input amount, it does not cause a serious inhibitory effect due to excessive substrates, as obtained from Examples 1, 6-7.
[0105] The HPLC spectrum of vanillin detection in Example 1 is as follows Figure 2 As shown;
[0106] Embodiment 8-13
[0107] The corresponding relationship of the preparation method of producing isoeugenol vanillin by using Amycolatopsis spp. is shown in the following table.
[0108] Table: Comparison table of the use of isoeugenol vanillin produced by using Amycolatopsis in Examples 8-13
[0109] Group Production of isoeugenol vanillin by using Amycolatopsis spp. Example 8 Prepared from Preparation Example 15 Example 9 Prepared from Preparation Example 16 Example 10 Prepared from Preparation Example 17 Embodiment 11 Prepared from Preparation Example 18 Example 12 Prepared from Preparation Example 19 Example 13 Prepared from Preparation Example 20
[0110] The isoeugenol vanillin produced by using Amycolatopsis sp. in Examples 8-13 above was selected, and its yield, concentration and molar conversion rate were tested according to the above measurement steps. The average values of the test results were recorded in the following table.
[0111] Table: Performance test results of yield, concentration and molar conversion of Examples 1, 8-13
[0112]
[0113]
[0114] As can be seen from the above table, in the preparation process of isoeugenol vanillin produced by using Amycolatopsis in Examples 8-13, the effect of improving the production effect of isoeugenol vanillin produced by using Amycolatopsis is good, by using Amycolatopsis with non-toxic substances and low vanillin degradation rate as a platform. By integrating isoeugenol monooxygenase into the genome of Amycolatopsis strain, a microbial synthesis chassis of high-yield isoeugenol vanillin is constructed, thereby achieving the purpose of improving the production effect of isoeugenol vanillin produced by using Amycolatopsis;
[0115] Its yield is 80.92-82.03 g; its concentration is 26.97-27.34 mm / s; its molar conversion rate is 96.91-98.18%;
[0116] It can be seen that when the production raw materials are certain, the production effect of isoeugenol vanillin produced by Pseudomonas amycolata can be increased by adjusting the preparation conditions of the recombinant strain. It is not difficult to see from the data in the above table that when Pseudomonas amycolata is used to produce isoeugenol vanillin, when the ratio of the recombinant plasmid to Pseudomonas amycolata is 1:1, the vanillin yield is the highest. The reason for this is that this ratio can better balance the interaction between the plasmid and the bacteria; the number of bacteria and the amount of plasmid are adapted, so that the conversion process is relatively stable and efficient, which lays a good foundation for the subsequent fermentation of the transformed bacteria to produce vanillin, as obtained by Examples 1, 8-11.
[0117] It can be seen that when the production raw materials are certain, the production effect of isoeugenol vanillin produced by using Amycolatopsis can be increased by adjusting the preparation conditions of the recombinant strain. It is not difficult to see from the data in the above table that when Amycolatopsis is activated, the OD value after activation culture is increased, and the yield, concentration and molar conversion rate of isoeugenol vanillin produced by using Amycolatopsis are increased. The reason for this is that as the number of bacteria increases during the activation culture process, the metabolic activity of the entire cell is also improved. In order to maintain growth and reproduction, many metabolic pathways inside the bacteria are fully activated, the synthesis amount of various key enzymes involved in substrate conversion is increased, and the enzyme activity is also at a high level, which is obtained from Examples 1 and 12-13.
[0118] This specific embodiment is only an explanation of the present invention, not a limitation of the present invention. After reading this specification, those skilled in the art can make non-creative modifications to this embodiment as needed, but as long as it is within the scope of the claims of the present invention, it is protected by the patent law.
Claims
1. A method for producing isoeugenol vanillin using Amycolatopsis spp., characterized in that: The following steps are involved: A1. Take out the AsPnIEM glycerol bacteria recombinant strain from the low-temperature environment of -80℃, let it thaw naturally at room temperature, and after thawing, inoculate the strain on the surface of the activated solid culture medium and culture it at 30℃ for 3-4 days; use sterile tools to pick a single colony from the plate, inoculate it into the seed culture medium, and culture it at 30℃ and 220rpm for 24-48h until the OD 600 When the value reaches 7, seed solution is obtained; A2, inoculate the seed solution obtained in A1 into a 5L fermenter at a 10% inoculation rate, and place it in a 3L fermentation medium, adjust the pH to 7.2, and then ferment for 12-16h at a temperature of 30°C, a stirring speed of 500rpm, and a ventilation rate of 1-2vvm until the OD 600 The value reaches 3-4; at this time, a total of 90-150g of isoeugenol solution is added to the fermentation tank three times, and the fermentation conversion is continued for 50h to obtain the production of isoeugenol vanillin using Amycolatopsis spp.
2. A method for producing isoeugenol vanillin using Amycolatopsis according to claim 1, characterized in that: The recombinant strain is prepared from a recombinant plasmid and Amycolatopsis spp.
3. A method for producing isoeugenol vanillin using Amycolatopsis as claimed in claim 1, characterized in that: The raw material components and volume ratios of the recombinant strain are as follows: recombinant plasmid: Amycolatopsis is (1-3): (1-2).
4. A method for producing isoeugenol vanillin using Amycolatopsis according to claim 1, characterized in that: The preparation method of the recombinant strain comprises the following steps: C1. Place 1 μL of vdhL-F primer, 1 μL of vdhL-R primer, 1 μL of vdh gene DNA template, 10 μL of DNA polymerase, and 7 μL of water in a PCR instrument, pre-denature at 90°C for 5 min, then denature at 95°C for 30 s, then anneal at 55°C for 30 s, repeat the above operation for 35 cycles, amplify the upstream sequence of the vdh gene at 72°C and a speed of 1 kb / min to obtain the upstream sequence of the vdh gene, and then use the same conditions to amplify the downstream sequence of the vdh gene using vdhR-F primer and vdhR-R primer to obtain the downstream sequence of the vdh gene. C2. Mix 2 μL of pULcrRNA-km-Cas12 plasmid, 2 μL of PnIEM gene sequence, 2 μL of vdh gene upstream sequence, 2 μL of vdh gene downstream sequence, 2 μL of water and 10 μL of ligase and place in a PCR instrument. Perform seamless cloning reaction at 50°C for 30 minutes to obtain a recombinant plasmid named pULΔvdh:PnIEM. C3, taking out the glycerol bacteria of Pseudomonas amycolata stored in a -80°C refrigerator and thawing it at room temperature; then, inoculating the strain onto a solid culture medium for activation culture, and continuing to culture at a temperature of 30°C for 3 days; then, inoculating the strain into an activation culture medium, and continuing to culture at a temperature of 30°C and a rotation speed of 220 rpm for 2 days; when the OD value reaches 0.7-0.8, collecting the bacteria to obtain Pseudomonas amycolata; C4. After the recombinant plasmid pULΔvdh:PnIEM obtained in C2 is fully mixed with the Amycolatopsis pseudomycoticus obtained in C3, the mixture is electrotransformed under the conditions of an electric field strength of 20 kV / cm, a capacitance of 30 μF, a resistance of 300 Ω, and an electric shock time of 4 ms; then, the transformed cells are added to an activation medium and incubated at a temperature of 30°C and a rotation speed of 220 rpm for 24 hours. The incubated cells are spread on a solid culture medium containing 50 μg / ml apramycin and cultured at 30°C; after a single colony grows on the culture medium, a colony PCR test is performed using primers IEMcheck-F and IEMcheck-R to verify the correct insertion of the gene. After colony verification, the correct strain is confirmed to be added with glycerol and stored at -80°C for a long time to obtain a recombinant strain, which is named AsPnIEM.
5. The method for producing isoeugenol vanillin using Amycolatopsis according to claim 1, characterized in that: The formula of the solid culture medium is: 4 g / L glucose, 4 g / L yeast extract, 10 g / L malt extract powder, and 20 g / L agar powder.
6. A method for producing isoeugenol vanillin using Amycolatopsis according to claim 4, characterized in that: The formula of the activation culture medium is: 10 g / L glucose, 10 g / L glycerol, 1 g / L beef extract, 2 g / L peptone, and 1 g / L yeast extract.
7. The method for producing isoeugenol vanillin using Amycolatopsis according to claim 1, characterized in that: The activated solid culture medium has a formula of: 10 g / L glucose, 10 g / L glycerol, 1 g / L beef extract, 2 g / L peptone, 1 g / L yeast extract, and 20 g / agar powder.
8. The method for producing isoeugenol vanillin using Amycolatopsis according to claim 1, characterized in that: The fermentation medium formula is: sucrose 103g / L, yeast extract 1.5g / L, Na2HPO4 4g / L, NaCl 0.2g / L, KH2PO4 0.1g / L, MgSO4·7H2O 0.2g / L, CaCl2 0.05g / L.
9. The method for producing isoeugenol vanillin using Amycolatopsis according to claim 1, characterized in that: The seed culture medium formula is: glucose 5g / L, yeast extract 10g / L, KH2PO4 1g / L, Na2HPO4 4g / L, MgSO4·7H2O 0.2g / L, NaCl 0.2g / L, CaCl2 0.05 g / L.