Application of pichia guilliermondii-bacillus aryabhattai co-fermentation in preparation of dopamine and specific method
Through the co-fermentation system of Pichia jiyemon GXDK6 and Bacillus adenus NM1-A2, dopamine was fermented in one step using cheap carbon sources to ferment biosynthetic dopamine, which solved the problems of low yield and high cost of dopamine synthesis, and achieved efficient and stable dopamine production.
Patent Information
- Application Number
- CN202510182627.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-19
- Publication Date
- 2025-07-04
AI Technical Summary
The prior art is difficult to efficiently synthesize dopamine under low-cost conditions, the synthesis yield of a single strain is limited, the process is complex and easy to pollute the environment.
The co-fermentation system of Pichia citrus citrus meridian GXDK6 and Bacillus arrecitris NM1-A2 was adopted to use a cheap carbon source to biosynthesize dopamine, and the fermentation conditions were optimized to improve synthesis efficiency.
The dopamine concentration has reached 374.43 mg/L, and the synthesis is stable and repeatable, solving the problems of restricted source of dopamine and high production costs, and is suitable for large-scale production.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of microbiology, and relates to the application and specific method of Pichia guilliermondii GXDK6 and Bacillus aryabhattai NM1-A2 in the preparation of dopamine. Background Art
[0002] Dopamine is a neurotransmitter in the process of brain signal transmission and has significant curative effects in the treatment of diseases such as depression and myocardial infarction. In addition, dopamine also has the potential to be used as an innovative biomaterial in the production of lithium batteries, which can improve electrochemical performance and meet the high-power energy demands of electric vehicles and smart utility grids. In 2022, the global dopamine market revenue exceeded 320 million US dollars, with an annual growth rate of 8.2% in the next decade. Compared with chemical synthesis, microbial synthesis of dopamine has the advantages of good product naturalness, short fermentation cycle, and less environmental pollution, which is the development trend of future dopamine production. However, exploring microorganisms that can efficiently synthesize dopamine de novo under low-cost carbon source conditions remains a major challenge.
[0003] However, a single strain can only utilize specific metabolic pathways, resulting in limited synthesis yields. Co-fermentation can utilize various strains with different genotypes and metabolic characteristics to achieve metabolic sharing. This method reduces the complexity of substrate metabolism, inhibits the formation of by-products, accelerates the generation of products, and expands the scope of the biological reaction environment and metabolic pathway diversity. By controlling the type, quantity, and ratio of strains, co-fermentation can effectively regulate and optimize the fermentation metabolic pathway, thereby improving the process efficiency. Although some progress has been made in co-fermentation, the synthesis of natural metabolites by microbial co-fermentation remains challenging. Co-fermentation using yeast and Bacillus can solve the limitations of single-strain production of metabolites and provide valuable insights and references for the development of new and efficient biosynthetic strategies. Summary of the Invention
[0004] Aiming at the deficiencies of the prior art, the present invention provides a co-fermentation of Pichia guilliermondii GXDK6 and Bacillus aryabhattai NM1-A2 for dopamine synthesis, and the co-fermentation is a system that directly uses simple and inexpensive carbon sources for one-step fermentation biosynthesis of dopamine, which is discovered for the first time. The specific content is as follows:
[0005] Application of Pichia guilliermondii GXDK6 and Bacillus aryabhattai NM1-A2 in the preparation of dopamine. The Pichia guilliermondii GXDK6 and Bacillus aryabhattai NM1-A2 are taxonomically named Pichia guilliermondii GXDK6 and Bacillus aryabhattai NM1-A2, and are deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit numbers CGMCC No. 16007 and CGMCC No. 23142 respectively.
[0006] Furthermore, for the preparation of dopamine, the above co-fermentation system is used as the fermentation strain to prepare dopamine.
[0007] Furthermore, for the preparation of dopamine using the above co-fermentation system as the fermentation strain, specifically, Pichia guilliermondii GXDK6 and Bacillus aryabhattai NM1-A2 are inoculated and fermented, and then the fermentation broth is collected and concentrated, and then dopamine is extracted and separated to obtain its pure product.
[0008] Furthermore, the application in the preparation of dopamine is specifically that the above co-fermentation system is used as a bacterial preparation in fields such as agriculture, food, and pharmaceutical preparation that require the preparation of dopamine.
[0009] A method for biosynthesizing dopamine using the above co-fermentation system, using the microorganisms Pichia guilliermondii GXDK6 and Bacillus aryabhattai NM1-A2 as the starting strains for fermentation, and then collecting and concentrating the fermentation broth, and then extracting and separating dopamine to obtain its pure product.
[0010] Furthermore, this method uses the above co-culture system as the starting strain, uses inexpensive carbon sources (such as glucose, fructose, sucrose, etc.) and conventional culture medium components (peptone, yeast powder, potassium chloride) as the fermentation substrates, ferments under the conditions that the fermentation system is 100 - 5000 mL, the inoculation amount is 0.5% - 5.0%, the temperature is 30 - 37 °C, the pH is 4.0 - 10.0, and the stirring speed is 0 - 300 rpm for 16 - 120 h to produce dopamine.
[0011] Furthermore, the culture medium has a formula containing 2.0% glucose, 5.0% peptone, 5.0% yeast powder, a temperature of 37 °C, a pH of 7.0, a fermentation time of 24 h, and 1.5% NaCl.
[0012] Advantages of the present invention:
[0013] (1) The co-fermentation of Pichia guilliermondii GXDK6 and Bacillus aryabhattai NM1-A2 used in this application is the first discovered co-fermentation strain that directly uses simple and inexpensive carbon sources for one-step fermentation biosynthesis of dopamine.
[0014] (2) For Pichia guilliermondii GXDK6 used in this application, the concentration of biosynthesized dopamine reaches 374.43 mg / L, and the detection is stable with good repeatability. It solves key problems such as source limitation of dopamine, high production cost, complex process, and easy environmental pollution, and is expected to further scale up and industrialize the production of dopamine.
[0015] (3) The Pichia guilliermondii GXDK6 and Bacillus aryabhattai NM1-A2 strains used in this application have clear genetic backgrounds, stable activities, relatively fast growth rates, high safety, and simple culture conditions.
[0016] (4) Based on the fact that Pichia guilliermondii and Bacillus aryabhattai strains can efficiently biosynthesize dopamine, this application simultaneously optimized and explored the factors affecting dopamine synthesis (such as NaCl, pH) by controlling the single variable method, and obtained an excellent method for co-cultivation one-step fermentation biosynthesis of dopamine, laying a foundation for the industrialization of dopamine biosynthesis.
[0017] (5) Using the method of this application to prepare dopamine has the advantages of environmentally friendly production process, simple operation, low cost, and suitability for large-scale production. Description of the Drawings
[0018] Figure 1 It is based on gas chromatography-mass spectrometry to qualitatively analyze dopamine in co-fermentation.
[0019] Figure 2 It is the yield of dopamine under co-fermentation.
[0020] Figure 3 It is the yield of dopamine synthesized by co-cultivation fermentation under different peptone concentrations.
[0021] Figure 4 It is the yield of dopamine synthesized by co-cultivation fermentation under different yeast powder conditions.
[0022] Figure 5 It is the yield of dopamine synthesized by co-cultivation fermentation under different NaCl conditions. Detailed Embodiments
[0023] The following further elaborates on the present invention in detail with specific embodiments, which are explanations rather than limitations of the present invention.
[0024] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified.
[0025] All materials, reagents, etc. used in the following examples can be obtained commercially without special instructions.
[0026] Example 1 Activation of Pichia guilliermondii GXDK6 and Bacillus aryabhattai NM1-A2 and Preparation of Seed Liquids
[0027] The Pichia guilliermondii GXDK6 strain stored at -80°C was activated through a slant. The formula of the activation medium was: 2% glucose, 2% peptone, 2% agar powder, 1% yeast powder, 0.2% KH2PO4, and 0.2% CaCl2. The activation conditions were 35°C and the activation culture time was 2 - 3 days. After a single colony grew in the petri dish, an appropriate amount of the colony was picked and inoculated into 10 - 20 mL of a liquid medium (formula: 2.0% glucose, 2.0% peptone, 1.0% yeast powder, 0.2% KH2PO4, 0.2% CaCl2), and cultured with shaking at 35°C and 200 rpm to prepare the Pichia guilliermondii GXDK6 seed liquid.
[0028] The Bacillus aryabhattai NM1-A2 strain stored at -80°C was activated through a slant. The formula of the activation medium was: 1% peptone, 2% agar powder, 0.5% yeast powder, and 1% NaCl. The activation conditions were 37°C and the activation culture time was 1 day. After a single colony grew in the petri dish, an appropriate amount of the colony was picked and inoculated into 10 - 20 mL of a liquid medium (formula: 1% peptone, 0.5% yeast powder, 1% NaCl), and cultured with shaking at 37°C and 200 rpm to prepare the Bacillus aryabhattai NM1-A2 seed liquid.
[0029] Example 2 Qualitative Analysis of Dopamine Biosynthesized by the Co-culture System of Pichia guilliermondii GXDK6 and Bacillus aryabhattai NM1-A2 Using Gas Chromatography-Mass Spectrometry
[0030] (1) Biosynthesis of dopamine by the co-culture system through biological fermentation.
[0031] The seed liquids of Pichia guilliermondii GXDK6 and Bacillus aryabhattai NM1-A2 were inoculated into the fermentation medium (2.0% glucose, 2.0% peptone, 1.0% yeast powder, 1% NaCl) at a mixing ratio of 6:4 and an inoculation amount of 1% (V / V). The loading volume was 100 mL / 250 mL Erlenmeyer flask, the culture temperature was 37°C, the initial pH was in the natural state, and shaking culture was carried out at 200 rpm for 24 h. The fermentation broth was collected by centrifugation at 10000 rpm.
[0032] (2) Treatment of the fermentation broth sample.
[0033] 1) Take 1 mL of the bacterial solution for each sample under each condition and place it in a 1.5 mL centrifuge tube (in triplicate), centrifuge at 10,000 rpm for 10 min, and take the supernatant.
[0034] 2) Filter the supernatant with a 0.8 μm filter membrane and place it in a new 1.5 mL centrifuge tube.
[0035] 3) Take 500 μL of the above filtrate respectively and place it in a new 1.5 mL centrifuge tube, then add 10 μL of ribitol solution with a concentration of 0.1 mg / mL. Subsequently, all the samples after adding ribitol are frozen and centrifuged to concentrate to powder.
[0036] 4) Derivatization treatment of the sample. (Add 80 μL of 20 mg / mL methoxyamine hydrochloride - pyridine solution and react for 2 h)
[0037] 5) Alkylation treatment of the sample. (Add 80 μL of trichloroacetamide and react for 2 h, then centrifuge at 10,000 rpm for 10 min.)
[0038] 6) Gas chromatography - mass spectrometry (GC - MS) detection and analysis.
[0039] 7) Detection conditions of gas chromatography - mass spectrometry (GC / MS): Chemical analysis of the sample is carried out using a Thermo DSQII single quadrupole gas chromatography - mass spectrometry (GC / MS). The inlet temperature of the instrument is maintained at 250 °C. 1 μL of the derivatized sample is injected in splitless mode into a DBS (Dodecyl Benzene Sulfonic Acid) column (length 30 m × inner diameter 250 μm × thickness 0.25 μm). The source temperature of the mass spectrometer is maintained at 250 °C in the direct ionization mode with an ionization energy of 70 eV and an acceleration voltage of 8000 V. For the experiments of full scan and selected ion recording, the temperature of the mass spectrometer quadrupole is kept constant at 150 °C. The initial temperature of the gas chromatography oven is set at 85 °C for 5 minutes, then increased to 300 °C at a rate of 15 °C / min. The carrier gas of the gas chromatography is helium, and the flow rate is kept constant at 1 mL / min. The operating range of the mass spectrometry is 50 - 600 m / z.
[0040] The GC - MS detection results show that, compared with single - strain fermentation, the amount of dopamine synthesized in the co - culture system increases, and the results are as Figure 1 shown. Therefore, it is expected that the synthesis of dopamine by the co - fermentation of Pichia pastoris and Bacillus subtilis and its large - scale production will become a new source of dopamine.
[0041] Example 3 Qualitative and quantitative analysis of dopamine synthesized in the co - fermentation system under different mixing ratios using a high - performance liquid chromatograph
[0042] (1) Effects of the mixed ratio of *Pichia guilliermondii* GXDK6 and *Bacillus aryabhattai* NM1-A2 on dopamine synthesis.
[0043] The seed solutions of *Pichia guilliermondii* GXDK6 and *Bacillus aryabhattai* NM1-A2 were inoculated into the fermentation medium (2.0% glucose, 2.0% peptone, 1.0% yeast powder, 1.0% NaCl) at a mixed ratio of 0:10, 2:8, 4:6, 6:4, 8:2, 10:0 at an inoculation amount of 1% (V / V). The liquid loading was 100 mL / 250 mL Erlenmeyer flask, the culture temperature was 37 °C, the initial pH was natural, and the shaking culture was carried out at 200 rpm for 24 h. The fermentation broth was collected by centrifugation at 10000 rpm.
[0044] (2) Treatment of the fermentation broth samples.
[0045] 9 mL of the supernatant fermentation broth was taken from each bottle of fermentation broth and concentrated and dried by a centrifugal concentrator. After the concentration and drying were completed, 1 mL of ethanol was added to each tube of the sample, and after mixing evenly, it was impregnated overnight at 37 °C and 200 rpm, and then centrifuged at 12000 rpm for 10 min. After taking the supernatant, it was filtered through a 0.22 μm filter membrane and then subjected to liquid chromatography analysis.
[0046] (4) Detection conditions of high performance liquid chromatography (HPLC)
[0047] The high performance liquid chromatography (Agilent 1260 series) was equipped with an Innoval C18 chromatographic column (4.6 mm × 250 mm, 5 μm) and a DAD ultraviolet detector. Detection conditions: mobile phase 100% methanol: 1‰ phosphoric acid solution = 20:80, flow rate 0.8 mL / min, column temperature 30 °C, injection volume 10 μL, detection wavelengths were 220 nm and 280 nm.
[0048] The results were as Figure 2 It can be seen that different inoculation ratios in the co-fermentation will affect the dopamine content. With the increase of the inoculation ratio, the dopamine content first increases and then decreases. When the inoculation ratio is 6:4, the dopamine content is the highest within 24 hours, which is 374.43 mg / L. The dopamine synthesized by co-fermentation is 2.89 times and 1.74 times that of GXDK6 and NM1-A2 respectively, indicating that the co-fermentation system improves the synthesis yield of dopamine.
[0049] Example 4 Quantitative determination of the dopamine content in co-fermentation under different NaCl conditions by high performance liquid chromatography
[0050] (1) Synthesis of dopamine by co-fermentation.
[0051] The seed solutions of Pichia guilliermondii GXDK6 and Bacillus aryabhattai NM1-A2 were mixed at a ratio of 6:4 and inoculated into the fermentation medium containing 0.5%, 1.0%, 1.5%, 2.0%, and 2.5% NaCl (2.0% glucose, 2.0% peptone, 1.0% yeast powder, 1% NaCl) at an inoculation amount of 1% (V / V). The liquid loading was 100 mL / 250 mL Erlenmeyer flask, the culture temperature was 37 °C, the initial pH was in the natural state, and the shaking culture was carried out at 200 rpm for 24 h. The fermentation broth was collected by centrifugation at 10,000 rpm.
[0052] (2) Treatment of the fermentation broth sample.
[0053] 9 mL of the supernatant fermentation broth was taken from each bottle of fermentation broth and concentrated and dried using a centrifugal concentrator. After the concentration and drying were completed, 1 mL of ethanol was added to each tube of the sample, and the mixture was evenly mixed and impregnated overnight at 37 °C and 200 rpm. Then, it was centrifuged at 12,000 rpm for 10 min. After taking the supernatant, it was filtered using a 0.22 μm filter membrane and then subjected to liquid chromatography analysis.
[0054] (3) HPLC detection conditions
[0055] The high-performance liquid chromatograph (Agilent 1260 series) was equipped with an Innoval C18 chromatographic column (4.6 mm × 250 mm, 5 μm) and a DAD ultraviolet detector. Detection conditions: mobile phase 100% methanol: 1‰ phosphoric acid solution = 20:80, flow rate 0.8 mL / min, column temperature 30 °C, injection volume 10 μL, detection wavelengths were 220 nm and 280 nm.
[0056] The results showed that as the NaCl content increased (from 0.5% to 2.5.0%), the dopamine content in Pichia guilliermondii first increased and then decreased, and the dopamine content at 1.50% was 1.05 times that under the original culture conditions. As Figure 3 shown.
[0057] Example 5 Quantitative determination of the dopamine content in co-fermentation under different peptone conditions using a high-performance liquid chromatograph
[0058] (1) Co-culture biological fermentation synthesis of dopamine.
[0059] The seed solutions of Pichia guilliermondii GXDK6 and Bacillus aryabhattai NM1-A2 were mixed at a ratio of 6:4 and inoculated into the fermentation medium containing 1.0%, 2.0%, 3.0%, 4.0%, and 5.0% peptone respectively at an inoculation amount of 1% (V / V) (2.0% glucose, 2.0% peptone, 1.0% yeast powder, 1% NaCl). The liquid loading was 100 mL / 250 mL Erlenmeyer flask, the culture temperature was 37 °C, the initial pH was natural, and the shaking culture was carried out at 200 rpm for 24 h. The fermentation broth was collected by centrifugation at 10000 rpm.
[0060] (2) Treatment of the fermentation broth sample.
[0061] 9 mL of the supernatant fermentation broth was taken from each bottle of fermentation broth and concentrated and dried by a centrifugal concentrator. After the concentration and drying were completed, 1 mL of ethanol was added to each tube of the sample, and after mixing evenly, it was impregnated overnight at 37 °C and 200 rpm, then centrifuged at 12000 rpm for 10 min. After taking the supernatant, it was filtered through a 0.22 μm filter membrane and then subjected to liquid chromatography analysis.
[0062] (3) HPLC detection conditions
[0063] The high-performance liquid chromatograph (Agilent 1260 series) was equipped with an Innoval C18 chromatographic column (4.6 mm × 250 mm, 5 μm) and a DAD ultraviolet detector. Detection conditions: mobile phase 100% methanol: 1‰ phosphoric acid solution = 20:80, flow rate 0.8 mL / min, column temperature 30 °C, injection volume 10 μL, detection wavelengths 220 nm and 280 nm.
[0064] The results showed that as the peptone concentration increased (1% - 5%), the dopamine content showed an upward trend, and the content was the highest at 5%, which was 1.80 times that under the original culture conditions ( Figure 4 ), indicating that the peptone content had an impact on the synthesis of dopamine.
[0065] Example 6 Quantitative determination of dopamine content in co-fermentation under different yeast powder conditions using a high-performance liquid chromatograph
[0066] (1) Co-culture biological fermentation to synthesize dopamine.
[0067] The seed liquid of Pichia guilliermondii GXDK6 and Bacillus arnoldii NM1-A2 was mixed in a ratio of 6:4 and inoculated at an inoculation amount of 1% (V / V) into a fermentation medium containing 1.0%, 2.0%, 3.0%, 4.0%, and 5.0% yeast powder (2.0% glucose, 2.0% peptone, 1.0% yeast powder, 1% NaCl), the liquid volume was 100mL / 250mL Erlenmeyer flask, the culture temperature was 37°C, the initial pH was natural, and the shaking culture was carried out at a speed of 200rpm for 24h, and the fermentation liquid was collected by centrifugation at 10000rpm.
[0068] (2) Processing of fermentation broth samples.
[0069] 9 mL of supernatant fermentation liquid was drawn from each bottle of fermentation liquid and concentrated and dried using a centrifugal concentrator. After concentration and drying, 1 mL of ethanol was added to each tube of sample, mixed evenly, and immersed overnight at 37°C and 200 rpm, then centrifuged at 12,000 rpm for 10 min, and the supernatant was filtered using a 0.22 μm filter membrane and analyzed by liquid chromatography.
[0070] (3) HPLC detection conditions
[0071] The high performance liquid chromatograph (Agilent 1260 series) was equipped with an Innova! C18 column (4.6 mm × 250 mm, 5 μm) and a DAD UV detector. Detection conditions: mobile phase 100% methanol: 1‰ phosphoric acid solution = 20:80, flow rate 0.8 mL / min, column temperature 30°C, injection volume 10 μL, detection wavelengths 220 nm and 280 nm.
[0072] The results showed that with the increase of yeast powder concentration (1%-5%), the dopamine content showed an upward trend and reached the maximum value at 5%, which was 2.84 times that of the original culture conditions ( Figure 5 ), indicating that yeast powder can significantly affect the synthesis of dopamine.
[0073] The above tests show that the mixed system can be used as a fermentation strain to prepare and produce dopamine and is suitable for popularization and use.
[0074] Finally, it should be pointed out that the above embodiments are only representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.
Claims
1. Application of Pichia guilliermondii GXDK6 and Bacillus aryabhattai NM1-A2 in the preparation of dopamine.
2. The application according to claim 1, wherein The Pichia guilliermondii GXDK6 and Bacillus aryabhattai are respectively taxonomically named Pichia guilliermondii GXDK6 and Bacillus aryabhattai NM1-A2, and are deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit numbers CGMCC No. 16007 and CGMCC No. 23142 respectively.
3. The application according to claim 1, wherein The preparation of dopamine is to ferment and prepare dopamine with the co-culture system of Pichia guilliermondii GXDK6 and Bacillus aryabhattai NM1-A2.
4. The application according to claim 3, characterized in that, Using the above two microorganisms as fermentation strains to prepare dopamine, specifically, inoculate and ferment the co-culture system, then collect and concentrate the fermentation broth, and then extract and separate dopamine to obtain its pure product.
5. The application according to claim 1, characterized in that, The application in the preparation of dopamine is specifically to use the above two strains as bacterial preparations in fields such as agriculture, food, and pharmaceutical preparation that require the preparation of dopamine.
6. A method for biosynthesizing dopamine by co-fermentation of Pichia guilliermondii GXDK6 and Bacillus aryabhattai NM1-A2, characterized in that, Using the microorganisms Pichia guilliermondii GXDK6 and Bacillus aryabhattai NM1-A2 as starting strains for fermentation, then collect and concentrate the fermentation broth, and then extract and separate dopamine to obtain its pure product.
7. The method for synthesizing dopamine by biological fermentation using Pichia guilliermondii GXDK6 according to claim 6, wherein Using the microorganisms Pichia guilliermondii GXDK6 and Bacillus aryabhattai NM1-A2 as starting strains, using carbon sources and conventional culture medium components as fermentation substrates, ferment for 16 - 120 h under the conditions that the fermentation system is 100 - 5000 mL, the inoculum size is 0.5% - 5.0%, the temperature is 30 - 37 °C, the pH is 4.0 - 10.0, and the stirring speed is 0 - 300 rpm to produce dopamine.
8. The method for biosynthesizing dopamine by using Pichia guilliermondii GXDK6 according to claim 7, characterized in that, The carbon source is one or a mixture of glucose, fructose, and sucrose.
9. The method for biosynthesizing dopamine by using Pichia guilliermondii GXDK6 according to claim 7, characterized in that, The conventional culture medium components contain peptone, yeast powder, and potassium chloride.
10. The method for synthesizing dopamine by biological fermentation using Pichia guilliermondii GXDK6 according to claim 7, characterized in that, The culture medium has a formula containing 2.0% glucose, 5.0% peptone, 5.0% yeast powder, a temperature of 37 °C, a pH of 7.0, a fermentation time of 24 h, and 1.5% NaCl.