Method for producing isoeugenol vanillin by using pichia pastoris

Abstract

The invention relates to the technical field of biological preparation, and discloses a method for producing isoeugenol vanillin by using pichia pastoris, which comprises the following preparation method: 1, constructing a strain, obtaining an amino acid sequence of Pn I EM (C3VA26) from an NCBI database, carrying out gene optimization and synthesis on the amino acid sequence, amplifying an upstream sequence of a Pn I EM gene by using specific primers I EM-F and I EM-R, and carrying out gene amplification on the upstream sequence of the Pn I EM gene by using specific primers I EM-F and I EM-R; simultaneously amplifying the pPIC9K plasmid by using a 9K-F primer and a 9K-R primer; then, by means of a seamless cloning technology, the two gene sequences are accurately spliced, and the plasmid pPIC9K-Pn I EM is successfully constructed. According to the method for producing the isoeugenol vanillin by utilizing the pichia pastoris, the isoeugenol vanillin can be prepared through a biotechnological means, the preparation of the vanillin can be greatly improved, the purity is relatively high, the manufacturing cost is relatively low, the environment is not easy to pollute, the vanillin is prepared through a biological method, and the method is suitable for industrial production. Therefore, efficient, natural and industrial preparation of vanillin is realized.

Description

Technical Field

[0001] The invention relates to the technical field of biological preparation, in particular to a method for producing isoeugenol vanillin by utilizing Pichia pastoris. Background Art

[0002] Vanillin, also known as vanillin, is the first synthetic flavor in human history. This milestone achievement is attributed to the contribution of scientists in 1874. As a substance widely used in food additives, vanillin can significantly enhance the milky flavor of food and is commonly found in desserts such as cakes and chocolates. In addition, it is also widely used in daily chemical products, medicine and other fields. This flavor can be found in nature, especially in vanilla beans, and can also be produced through artificial synthesis. At present, vanillin is one of the most produced synthetic flavors in the world.

[0003] Isoeugenol is one of the key raw materials for scented tea production, which can be converted into vanillin through chemical reactions or biotechnology. Although chemical synthesis can produce vanillin, its yield is low, resulting in low resource utilization, and may produce more by-products during the production process, which will affect the purity of the final product.

[0004] In addition, chemical synthesis methods usually require the use of toxic and hazardous chemical raw materials and solvents, which not only increases production costs but also may pollute the environment. Due to the high cost of raw materials and the complexity of the synthesis process, the overall production cost is also relatively high.

[0005] Therefore, in practical applications, chemical synthesis of vanillin has certain limitations. In view of this, people prefer to use biological methods to synthesize vanillin, such as using microorganisms or enzyme catalysis technology to efficiently synthesize natural vanillin from isoeugenol.

[0006] This study utilized this simple metabolic pathway of Pichia pastoris strain GS115, recombined and introduced the enzyme that converts isoeugenol to vanillin into its genome, thereby realizing the biological preparation of vanillin, which marks the possibility of efficient and natural industrial conversion of vanillin. Summary of the invention

[0007] The invention provides a method for producing isoeugenol vanillin by utilizing Pichia pastoris, which can produce vanillin through industrial conversion.

[0008] The present invention provides the following technical solution: a method for producing isoeugenol vanillin using Pichia pastoris, comprising the following preparation method:

[0009] Step 1: Construction of strains

[0010] The amino acid sequence of PnIEM (C3VA26) was obtained from the NCBI database, optimized and synthesized, and the upstream sequence of the PnIEM gene was amplified using specific primers IEM-F and IEM-R, while the pPIC9K plasmid was amplified using primers 9K-F and 9K-R.

[0011] Then, using seamless cloning technology, the two gene sequences were precisely spliced ​​together to successfully construct the plasmid pPIC9K-PnIEM;

[0012] Take out the stored DH5α competent cells from the -80℃ freezer and thaw them in an ice box. Use the heat shock method to transfer the plasmid pPIC9K-PnIEM into DH5α, use LB solid medium containing ampicillin for screening, use primers 5AOX and 3AOX for colony PCR detection to verify the gene, and after colony verification, confirm that the correct strain is added with glycerol for long-term storage at -80℃ and named DH5α9KPnIEM;

[0013] The frozen strain DH5α9KPnIEM was taken out from the -80°C refrigerator, inoculated into LB liquid medium containing ampicillin, and cultured overnight at 37°C and 220 rpm; the bacteria were collected by centrifugation at a speed of 8000 rpm for 5 minutes, the plasmid pPIC9K-PnIE was extracted using a kit, and the plasmid was digested with PmeⅠ enzyme. The system is as follows:

[0014] Components Sample volume PmeⅠ 2μl Buffer 23μl Plasmids 25μl

[0015] The reaction was carried out at 37°C for 3 h, and the fragments were recovered by isopropanol / ethanol precipitation and stored in a -20°C storage device for later use;

[0016] A single colony was picked from the newly activated YPD solid medium, inoculated into the YPD liquid medium, and cultured overnight in an apparatus at a temperature of 30°C and a rotation speed of 180 rpm; 0.1% of the overnight culture solution was inoculated into fresh YPD liquid medium, and cultured in an apparatus at a temperature of 30°C and a rotation speed of 150 rpm until OD 0.8; GS115 was transformed using the D0308 kit, and the transformed bacterial solution was coated on the MD solid medium;

[0017] The single clones on the MD solid culture medium were collected and resuspended in the YPD liquid culture medium, and then spread on the YPD solid culture medium containing 0.25 mg / ml, 0.50 mg / ml, 0.75 mg / ml, and 1 mg / ml G418; single clones were picked on the YPD solid culture medium with different concentrations of G418, inoculated into the YPD culture medium, resuscitated and cultured overnight at 30°C and 180 rpm, the culture solution was inoculated into the BMGY liquid culture medium, cultured overnight at 30°C-180 rpm, the bacteria were collected by centrifugation, the bacteria were washed with sterile water, the bacteria were resuspended in the BMMY liquid culture medium, cultured at 28°C-180 rpm, methanol solution was added every 24 hours, the supernatant was collected by centrifugation after 72 hours, and electrophoresis was performed. The single clone with the highest expression level was inoculated into the YPD liquid culture medium for culture, glycerol was added and stored in a -80°C refrigerator and named GS1159KPn I EM.

[0018] Step 2: Catalysis of the fermenter

[0019] The GS1159KPn I EM strain was taken out from a -80°C refrigerator, thawed at room temperature, inoculated into a triangular flask containing YPD liquid culture medium, and cultured overnight at 30°C-220rpm to obtain a resuscitation solution; the resuscitation solution was inoculated into a triangular flask containing BMGY liquid culture medium at a 5% inoculation amount and cultured at 30°C-220rpm to the logarithmic phase; the expansion culture solution was inoculated into a fermenter containing 2L of BMGY liquid culture medium at a ventilation volume of 0.5vvm, and the pH was adjusted to 8.5 using ammonia water and hydrochloric acid. When the glycerol in the culture medium was consumed, the dissolved oxygen rebounded, and glycerol was added in a stream. After the glycerol was replenished and the dissolved oxygen rebounded, 5ml of methanol was added. After the methanol addition was completed, 5ml of methanol and 20g of isoeugenol were added to carry out a catalytic reaction;

[0020] Step 3: Vanillin Extraction and Crystallization

[0021] The fermentation liquid was inactivated by heating, the pH was adjusted to 5 with HCl, and after centrifugation and sterilization, an equal amount of ethyl acetate was added for extraction twice; the organic phase was collected, and the organic phase was rotary evaporated at a temperature set at 40°C, with other conditions unchanged, and the concentration was stopped when crystals were about to precipitate in the concentrated liquid, and the evaporated ethyl acetate was recovered; two times the volume of n-hexane was added for crystallization;

[0022] Step 4: Vanillin detection

[0023] Weigh 2 g of crystalline vanillin and dissolve it in ethanol. After dilution, filter it with a 0.22 μM filter membrane and analyze it by HPLC. Use a C18 column, inject 10 μL, set the column temperature to 35°C, use 30% methanol + 0.1% acetic acid as the mobile phase, and the flow rate is 1 mL / min. The detection wavelength is 280 nm. The detection results show that the vanillin yield is 14 g.

[0024] Preferably, the culture medium is composed of the following components:

[0025] LB liquid medium: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L

[0026] LB solid medium: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, agar 15g / L LYPD liquid medium: peptone 20g / L, yeast powder 10g / L, glucose 20g / L

[0027] YPD solid medium: peptone 20g / L, yeast powder 10g / L, glucose 20g / L, agar powder 15g / L

[0028] MD solid medium: glucose 20g / L, yeast nitrogen base without amino acids (YNB) 13.4g / L, biotin 4mg / L, agar powder 15g / L

[0029] BMGY liquid medium: peptone 20g / L, yeast powder 10g / L, potassium phosphate 0.1M, YNB 13.4g / L, biotin 4mg / L, glycerol 20g / L

[0030] BMMY liquid culture medium: peptone 20 g / L, yeast powder 10 g / L, potassium phosphate 0.1 M, YNB 13.4 g / L, biotin 4 mg / L, methanol 3%.

[0031] Preferably, the preparation method involves a centrifuge, a constant temperature culture device and a frozen storage device.

[0032] The present invention has the following beneficial effects:

[0033] The method for producing isoeugenol vanillin by utilizing Pichia pastoris can prepare isoeugenol vanillin by biotechnology means, and the method can greatly improve the preparation of vanillin, and has high purity, low manufacturing cost, and is not prone to environmental pollution, thereby realizing the biological preparation of vanillin, which marks the realization of efficient and natural industrialized preparation of vanillin. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a molecular diagram of the synthetic pathway of isoeugenol vanillin of the present invention;

[0035] Figure 2 This is the plasmid map of pPIC9K-PnIEM of the present invention;

[0036] Figure 3 It is the SDS-PAGE electrophoresis diagram of the present invention;

[0037] Figure 4 The HPLC spectrum of vanillin detection of the present invention. DETAILED DESCRIPTION

[0038] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Please refer to Figure 1-4 , a method for producing isoeugenol vanillin using Pichia pastoris, comprising the following preparation method:

[0039] Step 1: Construction of strains

[0040] The amino acid sequence of PnIEM (C3VA26) was obtained from the NCBI database, optimized and synthesized, and the upstream sequence of the PnIEM gene was amplified using specific primers IEM-F and IEM-R, while the pPIC9K plasmid was amplified using primers 9K-F and 9K-R.

[0041] Then, using seamless cloning technology, the two gene sequences were precisely spliced ​​together to successfully construct the plasmid pPIC9K-PnIEM;

[0042] Take out the stored DH5α competent cells from the -80℃ freezer and thaw them in an ice box. Use the heat shock method to transfer the plasmid pPIC9K-PnIEM into DH5α, use LB solid medium containing ampicillin for screening, use primers 5AOX and 3AOX for colony PCR detection to verify the gene, and after colony verification, confirm that the correct strain is added with glycerol for long-term storage at -80℃ and named DH5α9KPnIEM;

[0043] The frozen strain DH5α9KPnIEM was taken out from the -80°C refrigerator, inoculated into LB liquid medium containing ampicillin, and cultured overnight at 37°C and 220 rpm; the bacteria were collected by centrifugation at a speed of 8000 rpm for 5 minutes, the plasmid pPIC9K-PnIE was extracted using a kit, and the plasmid was digested with PmeⅠ enzyme. The system is as follows:

[0044] Components Sample volume PmeⅠ 2μl Buffer 23μl Plasmids 25μl

[0045] The reaction was carried out at 37°C for 3 h, and the fragments were recovered by isopropanol / ethanol precipitation and stored in a -20°C storage device for later use;

[0046] A single colony was picked from the newly activated YPD solid medium, inoculated into the YPD liquid medium, and cultured overnight in an apparatus at a temperature of 30°C and a rotation speed of 180 rpm; 0.1% of the overnight culture solution was inoculated into fresh YPD liquid medium, and cultured in an apparatus at a temperature of 30°C and a rotation speed of 150 rpm until OD 0.8; GS115 was transformed using the D0308 kit, and the transformed bacterial solution was coated on the MD solid medium;

[0047] The single clones on the MD solid culture medium were collected and resuspended in the YPD liquid culture medium, and then spread on the YPD solid culture medium containing 0.25 mg / ml, 0.50 mg / ml, 0.75 mg / ml, and 1 mg / ml G418; single clones were picked on the YPD solid culture medium with different concentrations of G418, inoculated into the YPD culture medium, and resuscitated and cultured overnight at 30°C and 180 rpm. The culture solution was inoculated into the BMGY liquid culture medium, cultured overnight at 30°C-180 rpm, and the bacteria were collected by centrifugation. Sterile water was added to wash the bacteria, and the bacteria were resuspended in the BMMY liquid culture medium, cultured at 28°C-180 rpm, methanol solution was added every 24 hours, and the supernatant was collected by centrifugation after 72 hours for electrophoresis detection. The single clone with the highest expression level was inoculated into the YPD liquid culture medium for culture, and glycerol was added to store it in a -80°C refrigerator and named GS1159KPnIEM.

[0048] Step 2: Catalysis of the fermenter

[0049] Take out the GS1159KPnIEM strain from a -80°C refrigerator, thaw at room temperature, inoculate in a triangular flask filled with YPD liquid culture medium, and culture at 30°C-220rpm overnight to obtain a resuscitation solution; inoculate the resuscitation solution into a triangular flask filled with BMGY liquid culture medium at a 5% inoculation amount and culture at 30°C-220rpm to the logarithmic phase; inoculate the expansion culture solution into a fermenter filled with 2L of BMGY liquid culture medium at a ventilation volume of 0.5vvm, adjust the pH to 8.5 using ammonia water and hydrochloric acid, and when the glycerol in the culture medium is consumed, the dissolved oxygen rebounds, and then start to add glycerol, and after the glycerol is replenished and the dissolved oxygen rebounds, start to add 5ml of methanol, and after the addition of methanol is completed, add 5ml of methanol and 20g of isoeugenol to carry out a catalytic reaction;

[0050] Step 3: Vanillin Extraction and Crystallization

[0051] The fermentation liquid was inactivated by heating, the pH was adjusted to 5 with HCl, and after centrifugation and sterilization, an equal amount of ethyl acetate was added for extraction twice; the organic phase was collected, and the organic phase was rotary evaporated at a temperature set at 40°C, with other conditions unchanged, and the concentration was stopped when crystals were about to precipitate in the concentrated liquid, and the evaporated ethyl acetate was recovered; two times the volume of n-hexane was added for crystallization;

[0052] Step 4: Vanillin detection

[0053] Weigh 2 g of crystalline vanillin and dissolve it in ethanol. After dilution, filter it with a 0.22 μM filter membrane and analyze it by HPLC. Use a C18 column, inject 10 μL, set the column temperature to 35°C, use 30% methanol + 0.1% acetic acid as the mobile phase, and the flow rate is 1 mL / min. The detection wavelength is 280 nm. The detection results show that the vanillin yield is 14 g.

[0054] Experimental strain description:

[0055]

[0056] Primer ingredients description:

[0057]

[0058]

[0059] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for producing isoeugenol vanillin using Pichia pastoris, characterized in that: The invention comprises the following preparation methods: Step 1: Construction of strains The amino acid sequence of PnIEM (C3VA26) was obtained from the NCBI database, optimized and synthesized, and the upstream sequence of the PnIEM gene was amplified using specific primers IEM-F and IEM-R, while the pPIC9K plasmid was amplified using primers 9K-F and 9K-R. Then, using seamless cloning technology, the two gene sequences were precisely spliced ​​together to successfully construct the plasmid pPIC9K-PnIEM; Take out the stored DH5α competent cells from the -80℃ freezer and thaw them in an ice box. Use the heat shock method to transfer the plasmid pPIC9K-PnIEM into DH5α, use LB solid medium containing ampicillin for screening, use primers 5AOX and 3AOX for colony PCR detection to verify the gene, and after colony verification, confirm that the correct strain is added with glycerol for long-term storage at -80℃ and named DH5α9KPnIEM; The frozen strain DH5α9KPnIEM was taken out from the -80°C refrigerator, inoculated into LB liquid medium containing ampicillin, and cultured overnight at 37°C and 220 rpm; the bacteria were collected by centrifugation at a speed of 8000 rpm for 5 minutes, the plasmid pPIC9K-PnIE was extracted using a kit, and the plasmid was digested with PmeⅠ enzyme. The system is as follows: The reaction was carried out at 37°C for 3 h, and the fragments were recovered by isopropanol / ethanol precipitation and stored in a -20°C storage device for later use; A single colony was picked from the newly activated YPD solid medium, inoculated into the YPD liquid medium, and cultured overnight in an apparatus at a temperature of 30°C and a rotation speed of 180 rpm; 0.1% of the overnight culture solution was inoculated into fresh YPD liquid medium, and cultured in an apparatus at a temperature of 30°C and a rotation speed of 150 rpm until OD 0.8; GS115 was transformed using the D0308 kit, and the transformed bacterial solution was coated on the MD solid medium; The single clones on the MD solid culture medium were collected and resuspended in the YPD liquid culture medium, and then spread on the YPD solid culture medium containing 0.25 mg / ml, 0.50 mg / ml, 0.75 mg / ml, and 1 mg / ml G418; single clones were picked on the YPD solid culture medium with different concentrations of G418, inoculated into the YPD culture medium, and resuscitated and cultured overnight at 30°C and 180 rpm. The culture solution was inoculated into the BMGY liquid culture medium, cultured overnight at 30°C-180 rpm, and the bacteria were collected by centrifugation. Sterile water was added to wash the bacteria, and the bacteria were resuspended in the BMMY liquid culture medium, cultured at 28°C-180 rpm, methanol solution was added every 24 hours, and the supernatant was collected by centrifugation after 72 hours for electrophoresis detection. The single clone with the highest expression level was inoculated into the YPD liquid culture medium for culture, and glycerol was added to store it in a -80°C refrigerator and named GS1159KPnIEM. Step 2: Catalysis of the fermenter Take out the GS1159KPnIEM strain from a -80°C refrigerator, thaw at room temperature, inoculate in a triangular flask filled with YPD liquid culture medium, and culture at 30°C-220rpm overnight to obtain a resuscitation solution; inoculate the resuscitation solution into a triangular flask filled with BMGY liquid culture medium at a 5% inoculation amount and culture at 30°C-220rpm to the logarithmic phase; inoculate the expansion culture solution into a fermenter filled with 2L of BMGY liquid culture medium at a ventilation volume of 0.5vvm, adjust the pH to 8.5 using ammonia water and hydrochloric acid, and when the glycerol in the culture medium is consumed, the dissolved oxygen rebounds, and then start to add glycerol, and after the glycerol is replenished and the dissolved oxygen rebounds, start to add 5ml of methanol, and after the addition of methanol is completed, add 5ml of methanol and 20g of isoeugenol to carry out a catalytic reaction; Step 3: Vanillin Extraction and Crystallization The fermentation broth was inactivated by heating, adjusted to pH 5 with HCl, sterilized by centrifugation, and extracted twice with an equal amount of ethyl acetate; Collect the organic phase, and perform rotary evaporation of the organic phase. Set the temperature at 40°C, and keep other conditions unchanged. Stop concentration when crystals are about to precipitate in the concentrated solution, and recover the evaporated ethyl acetate; add twice the volume of n-hexane for crystallization; Step 4: Vanillin detection Weigh 2 g of crystalline vanillin and dissolve it in ethanol. After dilution, filter it with a 0.22 μM filter membrane and analyze it by HPLC. Use a C18 column, inject 10 μL, set the column temperature to 35°C, use 30% methanol + 0.1% acetic acid as the mobile phase, and the flow rate is 1 mL / min. The detection wavelength is 280 nm. The detection results show that the vanillin yield is 14 g.

2. A method for producing isoeugenol vanillin using Pichia pastoris according to claim 1, characterized in that: The culture medium is composed of the following components: LB liquid medium: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L LB solid medium: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, agar 15g / L YPD liquid medium: peptone 20g / L, yeast powder 10g / L, glucose 20g / L YPD solid medium: peptone 20g / L, yeast powder 10g / L, glucose 20g / L, agar powder 15g / L MD solid medium: glucose 20g / L, yeast nitrogen base without amino acids (YNB) 13.4g / L, biotin 4mg / L, agar powder 15g / L BMGY liquid medium: peptone 20g / L, yeast powder 10g / L, potassium phosphate 0.1M, YNB 13.4g / L, biotin 4mg / L, glycerol 20g / L BMMY liquid culture medium: peptone 20 g / L, yeast powder 10 g / L, potassium phosphate 0.1 M, YNB 13.4 g / L, biotin 4 mg / L, methanol 3%.

3. The method for producing isoeugenol vanillin using Pichia pastoris according to claim 1, characterized in that: The preparation method involves a centrifuge, a constant temperature culture device and a freezing storage device.

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