Method for electrosynthesis of 5-aminolevulinic acid by Pichia pastoris

Abstract

The present invention discloses a method for electrosynthesizing 5-aminolevulinic acid in Pichia pastoris. This method is based on constructing a reductive glycine pathway and a 5-ALA synthesis pathway in Pichia pastoris, and utilizes a microbial electrochemical system to provide external electrical energy to achieve the electrosynthesis of 5-ALA. The present invention uses Pichia pastoris as the research object. Previously, a highly efficient carbon-fixing reductive glycine pathway and a 5-ALA production pathway were constructed. Recombinant Pichia pastoris was applied to the microbial electrochemical system, using formic acid or CO2 as the carbon source and electrical energy supplied by the microbial electrochemical system as the energy source. The results investigate the production of 5-ALA in Pichia pastoris under electrical energy conditions.

Description

Technical Field

[0001] The present invention relates to the field of microbial electrochemistry, and in particular to a method for electrosynthesizing 5-aminolevulinic acid using Pichia pastoris. Background Art

[0002] Excessive CO2 emissions have led to a continuous increase in atmospheric CO2 concentrations, triggering a series of environmental problems, including the greenhouse effect and glacial melting. To mitigate the greenhouse effect, developing CO2 sequestration and utilization technologies has become a key research priority. Effective CO2 sequestration and utilization methods can transform waste CO2 into valuable resources, potentially mitigating rising atmospheric CO2 levels and the resulting ecological challenges.

[0003] Microbial CO2 fixation technology offers a promising new approach to achieving carbon neutrality. Through specific CO2 fixation pathways and energy input, microorganisms can convert CO2 into useful compounds under environmentally friendly conditions. The choice of CO2 fixation pathway and energy supply are key in the microbial CO2 fixation process. The reductive glycine pathway (RGP) is a biochemical process used for CO2 fixation in microorganisms. This pathway is an effective tool in CO2 fixation technology, particularly in environmental and industrial applications striving to achieve carbon neutrality. The RGP has attracted research attention for its efficient carbon fixation capacity and low energy consumption.

[0004] 5-Aminolevulinic acid (5-ALA) is a key non-protein amino acid widely used in medicine, agriculture, and the chemical industry. In medicine, 5-ALA is used as a photosensitizer in photodynamic therapy for certain cancers. Despite its wide range of uses, the production of 5-ALA faces challenges of high cost and low efficiency, particularly in biosynthesis. Microbial synthesis, while promising as an environmentally friendly approach to 5-ALA synthesis, is limited in commercial application by low yields. Among these biosynthetic methods, the use of yeast as a host for 5-ALA production faces a series of technical challenges. Yeast is widely used in biotechnology and has become a potential host for the synthesis of 5-ALA. However, yeast's natural porphyrin metabolic flux is weak, which means that its ability to synthesize 5-ALA in its unmodified state is limited.

[0005] As a methylotrophic microorganism, Pichia pastoris, due to its metabolic flexibility and industrial adaptability, is an ideal host for studying CO2 fixation and metabolic remodeling. By constructing a highly efficient carbon fixation and glycine reduction pathway and a 5-ALA synthesis pathway, Pichia pastoris achieves efficient CO2 fixation and 5-ALA synthesis under external electrical energy. This system design provides new technical support for the large-scale industrialization of CO2 fixation and conversion. Summary of the Invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to address the deficiencies of the existing technology and provide a method for electrosynthesizing 5-aminolevulinic acid (5-ALA) by Pichia pastoris. The method uses Pichia pastoris and a microbial electrochemical system to provide external electrical energy to achieve electrically driven synthesis of 5-ALA using formic acid or CO2.

[0007] In order to solve the above technical problems, the present invention discloses a method for electrosynthesizing 5-aminolevulinic acid in Pichia pastoris. The specific technical solution is as follows:

[0008] The present invention provides a method for electrosynthesizing 5-aminolevulinic acid using Pichia pastoris. The method comprises using an electrochemical device separated by a proton exchange membrane into a cathode chamber and an anode chamber, adding a catholyte to the cathode chamber and an anolyte to the anode chamber, and applying an external voltage to the electrochemical device to electrochemically synthesize 5-aminolevulinic acid (5-ALA). The catholyte includes a carbon source, a Pichia pastoris suspension, and an electron mediator; and the Pichia pastoris is a 5-aminolevulinic acid-producing Pichia pastoris.

[0009] The electron mediator is methyl viologen, and the Pichia pastoris is Pichia pastoris PMORG09-HEM1. The Pichia pastoris PMORG09-HEM1 is an autotrophic Pichia pastoris formed by constructing a methanol oxidation pathway, a glycine reduction pathway, a serine hydroxymethyl transfer pathway, and a 5-ALA synthesis pathway, detailed information of which has been disclosed in the reference [Guo, Yuanke, et al. Engineering yeasts to co-utilize methanol or formate coupled with CO2 fixation. Metabolic engineering 84:1-12.].

[0010] Wherein, in the cathode liquid, Pichia pastoris OD 600 is 30-50, and the concentration of the electron mediator is 0.15-0.8 mM. Preferably, the OD 600 The concentration of the electron mediator is 0.15 to 0.5 mM, more preferably 0.2 mM.

[0011] The Pichia pastoris resuspension is prepared by inoculating the activated Pichia pastoris strain into YPD liquid medium supplemented with an inducer, inducing the culture for 48 to 120 hours, and then resuspending the culture in an inorganic salt medium. The inducer is supplemented every 24 hours during the induction culture. Preferably, the inorganic salt medium comprises 2.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, and 0.5 g / L MgSO4·7H2O. The induction culture lasts for 72 hours.

[0012] The inducer is methanol; the initial addition concentration of the inducer is 0.8 to 1.2 g / L; and the amount of the inducer added each time during the induction culture is 0.8 to 1.2 g / L based on the volume of the initial YPD liquid culture medium. Preferably, the initial addition concentration of the inducer is 1 g / L, and the amount of the inducer added each time during the induction culture is 1 g / L based on the volume of the initial YPD liquid culture medium.

[0013] Wherein, the carbon source is any one of formic acid, formate or CO2 or a combination thereof, preferably any one of formic acid, formate or CO2.

[0014] When the carbon source is formic acid or formate, the initial concentration of the carbon source in the cathode liquid is 1 to 8 g / L. During the electrochemical synthesis process, the carbon source is added once every 12 to 24 hours, and the amount of the carbon source added each time is 2 to 3 g / L based on the volume of the initial cathode liquid. Preferably, the initial concentration of the carbon source in the cathode liquid is 3 g / L, and the carbon source is added once every 24 hours, and the amount of the carbon source added each time is 2 g / L based on the volume of the initial cathode liquid.

[0015] When the carbon source is CO2, different carbon source supply forms are involved. CO2 is added to the cathode liquid in the form of NaHCO3 and / or CO2 gas, wherein the concentration of NaHCO3 in the cathode liquid is 10-80 mM; CO2 gas is aerated in the cathode liquid at an aeration pressure of 1-5 kgf / cm 2 The aeration time is 2 to 5 minutes. During the electrochemical synthesis process, CO2 gas is added once every 8 to 24 hours. The aeration pressure of each CO2 gas addition is 5 kgf / cm 2 , the aeration time is 5min. Preferably, CO2 is added to the cathode liquid in the form of NaHCO3 and CO2 gas, the concentration of NaHCO3 in the cathode liquid is 50mM, and the aeration pressure of CO2 gas in the cathode liquid is 5kgf / cm 2 The aeration time is 5 minutes. During the electrochemical synthesis process, CO2 gas is added once every 12 hours. The aeration pressure of each addition is 5 kgf / cm 2 , the aeration time is 5 minutes.

[0016] The anolyte has a formula of: 2-3 g / L NaHPO4, 2-3 g / L NaH2PO4, 1-2 g / L NaCl, and 0.001-0.002 g / L dithiothreitol (DTT). Preferably, the formula is: 2.51 g / L NaHPO4·12H2O, 2.81 g / L NaH2PO4·2H2O, 1.45 g / L NaCl, and 0.002 g / L DTT.

[0017] The applied voltage provided is -0.8 to -1.2 V, preferably -0.8 V; and the electrochemical synthesis is carried out at 30°C.

[0018] Wherein, graphite felt is used as the electrode in the cathode chamber, and platinum sheet is used as the electrode in the anode chamber.

[0019] Beneficial effects:

[0020] Compared with the existing technology, the present invention provides a method for electrosynthesizing 5-aminolevulinic acid in Pichia pastoris, constructs an electrically driven 5-ALA electrochemical system, and regulates the growth environment of the recombinant Pichia pastoris by optimizing the concentration of electron mediators and electrochemical parameters within the electrochemical device. This allows the production of strains with improved 5-ALA production when using only formic acid or CO2 as the sole carbon source and external electrical energy as the energy source. Specific advantages are:

[0021] (1) The Pichia pastoris microbial electrochemical device constructed in this invention can use electricity as an energy source to drive microbial metabolic activity and directly synthesize target products through electricity, meeting the requirements of carbon neutrality and green production. Compared with chemical or solar energy supply, electricity can more precisely control the supply of electrons and the demand for reducing power, making it suitable for the continuous synthesis of high-value-added products such as 5-ALA, while reducing carbon source consumption and environmental pollution.

[0022] (2) The present invention utilizes high-density whole-cell catalysis to form 5-ALA, providing stronger catalytic efficiency and production rate, reducing the need for complex operations such as enzyme purification, and achieving sustainable green production. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0024] Figure 1 This is a structural diagram of the microbial electrochemical device.

[0025] Figure 2 This is a comparison chart of 5-ALA production by electric drive and non-electric drive.

[0026] Figure 3This is an analysis chart of 5-ALA production in the culture medium optimized with formic acid as the carbon source.

[0027] Figure 4 This is a graph showing the 5-ALA production analysis for the optimized induction time using formic acid as the carbon source. When the induction time is 72h, 96h, and 120h, the line graphs of formic acid concentration almost completely overlap.

[0028] Figure 5 Yield analysis plot for optimized formic acid supply concentration.

[0029] Figure 6 Yield analysis plot optimized for formic acid feeding.

[0030] Figure 7 This is the yield analysis diagram of voltage optimization using formic acid as the carbon source.

[0031] Figure 8 This is a yield analysis diagram for optimizing the ventilation frequency using CO2 as the only carbon source.

[0032] Figure 9 This is the yield analysis diagram for optimizing the electron mediator concentration with CO2 as the only carbon source.

[0033] Figure 10 CO2 is the only carbon source 13 C Tracer labeling diagram. DETAILED DESCRIPTION

[0034] The present invention is further illustrated below by describing specific implementation methods, but this is not intended to limit the present invention. Those skilled in the art can make various modifications or improvements based on the basic ideas of the present invention, but as long as they do not deviate from the basic ideas of the present invention, they are all within the scope of the present invention.

[0035] In the following examples, the YPD liquid culture medium has a formula of 20 g / L peptone, 10 g / L yeast powder, and 20 g / L glucose, and the solvent is pure water.

[0036] The MM culture medium is an inorganic salt culture medium, and its formula is: (NH4)2SO4 2.5g / L, KH2PO4 14.4g / L and MgSO4·7H2O 0.5g / L, and the solvent is pure water.

[0037] The formula of the anolyte is: NaHPO4·12H2O 2.51g / L, NaH2PO4·2H2O 2.81g / L, NaCl 1.45g / L, DTT 0.002g / L, pH 7.0, and the solvent is pure water.

[0038] The strain used in the present invention is Pichia pastoris PMORG09-HEM1, which is formed by constructing a methanol oxidation pathway, a glycine reduction pathway, a serine hydroxymethyl transfer pathway, and a 5-ALA synthesis pathway from autotrophic Pichia pastoris. The specific construction method is detailed in reference [Guo, Yuanke, et al. Engineering yeasts to co-utilize methanolor formate coupled with CO2 fixation. Metabolic engineering 84:1-12.].

[0039] In the following examples, the method for detecting 5-ALA production is as follows:

[0040] First, 5-ALA needs to be appropriately derivatized to enhance its volatility and thermal stability so as to meet the requirements of gas chromatography (GC). The specific method of the derivatization treatment is as follows: 2-3 mL of bacterial culture solution is collected, centrifuged at 8800 g at 4°C for 5 minutes, the bacteria are resuspended in 1 mL of pre-cooled ddH2O, and the bacteria are washed by centrifugation at 8800 g at 4°C for 5 minutes, and the mixture is repeated 2-3 times. 200 μL of 6M HCl is added, the bacteria are mixed, and the mixture is incubated at 99°C for 24 hours to lyse cellular proteins. After the incubation is completed, an equal volume of 6M NaOH is added to neutralize the HCl, and the mixture is centrifuged at 8800 g at 4°C for 1 minute. The supernatant is collected and lyophilized. The lyophilized sample is a sheet-like hollow stack, either white or light yellow, and is subjected to pre-column derivatization (derivation reagent is added according to the lyophilized volume. If the volume is too large, the amount of bacteria needs to be reduced. Standard samples are prepared by simultaneous derivatization). 50 μL of methoxyammonium hydrochloride / pyridine solution (20 mg / mL) is added to the lyophilized sample, vortexed to mix, and incubated in a 40°C water bath for 80 min; 80 μL of MSTFA (N-methyl-N-(trimethylsilyl)trifluoroacetamide) is added, vortexed to mix, and incubated in a 40°C water bath for 80 min; centrifuged at 10,000 g and 4°C for 5 min, and 100 μL of the supernatant is placed in an injection vial for GC-MS analysis.

[0041] High-resolution GC-MS (Thermo TRACE 1310 gas chromatograph, Orbitrap Exploris GC240 mass spectrometer, Thermo Scientific) was used to analyze the TM , USA) for the analysis of derivatized amino acid samples. The instrument was equipped with a TraceGOLD TG-5SilMS GC column (30 m × 0.25 mm, 0.25 μm film thickness, Thermo Scientific TM, USA). The oven temperature program was as follows: 60°C for 2 minutes, then ramped to 200°C at a rate of 5°C / min, then ramped to 320°C at a rate of 10°C / min and held for 3 minutes. Mass spectra of amino acids were acquired in the mass range of 50–650 m / z, with an acquisition rate of 5 spectra per second. The ion source and transfer line temperatures were 250°C and 290°C, respectively. Electron ionization was performed at 70 eV. Helium was used as the carrier gas at a constant flow rate of 1 mL / min. The sample injection volume was 1 μL, and each sample was run in triplicate. ThermoScientific FreeStyle 1.0 software was used for raw peak extraction, peak alignment, peak identification, and peak area integration.

[0042] Example 1 Construction of an electrically driven 5-ALA synthesis system

[0043] Pichia pastoris PMORG09-HEM1 strain was added to 10 mL YPD liquid medium and cultured overnight at 30°C and 200 rpm until OD 600 When the OD value reaches 2-3, the activated strain is obtained. 600 The cells were transferred to MM medium supplemented with methanol inducer (the concentration of methanol added was 1 g / L, where methanol can be used as both an inducer and a carbon source), placed in a 30°C shaker for culture, and methanol inducer was added at a cycle of 24 hours. The concentration of methanol added each time was 1 g / L (based on the volume of the initial MM medium). After 120 hours of shake flask induction culture, the cells were resuspended in MM medium supplemented with formic acid (the concentration of formic acid added was 3 g / L) to obtain a resuspension, and an electron mediator, methyl viologen, was added at a concentration of 0.5 mM. The methyl viologen was sterilized in the form of a methyl viologen aqueous solution through a membrane (0.22 μm) and then added to the resuspension. The initial OD value of the strain was 0. 600 40 aliquots were dispensed into the cathode chamber of the microbial electrochemical device. 50 mL of the anolyte solution was then poured into the anode chamber of the microbial electrochemical device. A voltage (-0.8 V) was applied to one group, while no voltage was applied to the other group. 5-ALA production was monitored over a 24-hour period.

[0044] The cathode chamber and the anode chamber are separated by a proton exchange membrane. The material of the anode (counter electrode) is a platinum sheet, the material of the cathode (working electrode) is a graphite felt, the reference electrode is an AgCl / Ag electrode, and the remaining components and connection methods are conventional means in this field. The constructed electrochemical device is placed in 75% alcohol and allowed to stand overnight. The electrochemical device is taken out after soaking, placed in a clean bench, irradiated with an ultraviolet lamp and blown dry. The structure diagram of the electrochemical device is shown in FIG. Figure 1 shown.

[0045] Electric energy drive output analysis chart as follows Figure 2 As shown. Figure 2It can be seen that by introducing the 5-ALA synthesis pathway into Pichia pastoris, electrical energy drives the Pichia pastoris glycine reduction system to synthesize 5-ALA (the yield can reach 20 mg / L), and the 5-ALA yield of the electrosynthesis system is twice as high as that of the control system without applying voltage.

[0046] This example demonstrates that Pichia pastoris PMORG09-HEM1 can successfully achieve electrically driven synthesis of 5-ALA.

[0047] Example 2 Optimization of culture medium

[0048] Following the method described in Example 1, an electrically driven glycine reduction system was constructed to synthesize 5-ALA. The MM medium during the induction period described in Example 1 was replaced with YPD medium, while other conditions remained unchanged. The 5-ALA yield was monitored over a 24-hour period. The results showed that replacing the MM medium with YPD medium increased the yield by more than two times, from 20 mg / L to 50.9 mg / L. Figure 3 This may be because YPD medium provides more nutrients and growth factors. Therefore, YPD medium was selected as the induction medium in the following experiments.

[0049] Example 3 Optimization of methanol induction time

[0050] According to the method described in Example 2, an electric energy driven reduction system for glycine was constructed to synthesize 5-ALA. The induction time of adding methanol inducer to the shake flask was 48h, 72h, 96h and 120h, and the other conditions remained unchanged. The 5-ALA production was monitored at a cycle of 24h. The results are shown in Figure 2. Figure 4 shown.

[0051] Depend on Figure 4 As can be seen, 5-ALA production was similar at 72, 96, and 120 h of induction, while 5-ALA production was lower at 48 h of induction. Considering time and economic costs, subsequent experiments chose to perform whole-cell electrical-driven 5-ALA synthesis in high-density cells after 72 h of induction.

[0052] Example 4 Optimization of formic acid concentration

[0053] According to the method of Example 3, an electric energy driven glycine reduction system with appropriate induction time was constructed to synthesize 5-ALA. The formic acid concentration was changed to 3g / L, 5g / L, and 8g / L, while the other conditions remained unchanged. The 5-ALA production was monitored over a 24-hour period. The results are shown in Figure 3. Figure 5 shown.

[0054] from Figure 5As can be seen, the higher the formic acid concentration, the stronger the inhibitory effect. A formic acid concentration of 3 g / L is just sufficient for Pichia pastoris catalysis, achieving nearly 100% formic acid consumption and 5-ALA production of 51 mg / L. Therefore, it can be concluded that a formic acid concentration of 3 g / L is optimal for Pichia pastoris electrocatalytic synthesis of 5-ALA.

[0055] Given that 5-ALA production was still very low and formic acid was almost completely utilized, we considered whether there was insufficient formic acid supply. Therefore, we added 2 g / L formic acid feed (based on the initial catholyte volume) at 24 h and 48 h.

[0056] from Figure 6 It can be seen that adding formic acid feed at 24h and 48h can indeed increase the electrocatalytic 5-ALA production, reaching 60.9mg / L.

[0057] Example 5 Optimization of applied voltage

[0058] Following the method of Example 4, a voltage-dependent electrochemical reduction system for glycine was constructed to synthesize 5-ALA. The applied voltages were -0.8 V, -1.2 V, and -1.5 V. All other conditions remained unchanged. 5-ALA production was monitored over a 24-hour period.

[0059] Depend on Figure 7 It can be seen that the higher the negative voltage applied, the lower the yield and the greater the inhibitory effect. The highest 5-ALA yield is achieved at -0.8V.

[0060] Example 6: Exploration of the yield of 5-ALA synthesized by electrocatalysis using CO2 as the only carbon source

[0061] Pick the positive clone of strain PMORG09-HEM1 and transfer it to 10 mL YPD liquid medium. Culture it overnight at 30°C and 200 rpm until the OD 600 When the OD value reaches 2-3, the activated strain is obtained. 600 0.2 was transferred to YPD medium supplemented with methanol inducer (the concentration of methanol added was 1 g / L), placed in a 30°C shaker for culture, and methanol inducer was added every 24 h. The concentration of methanol added each time was 1 g / L (based on the volume of the initial YPD liquid medium. After 72 h of shake flask culture, the cells were resuspended in MM medium to obtain a resuspension, and electron mediators methyl viologen and NaHCO3 were added. The concentrations of methyl viologen and NaHCO3 were 0.5 mM and 50 mM, respectively. The methyl viologen and NaHCO3 were sterilized in the form of an aqueous solution through a membrane (0.22 μm) and then added to the resuspension. The initial OD value of the strain was 0. 60040 points were filled into the cathode chamber of the microbial electrochemical device. 50 mL of the anolyte solution was poured into the anode chamber of the microbial electrochemical device, and one end of the vent tube with a 0.2 μm microporous filter membrane was connected to a CO2 gas cylinder (the CO2 ventilation pressure was 5 kgf / cm 2 , ventilation time is 5min), and the cathode solution is introduced into the other end. A voltage of -0.8V is applied. 5-ALA production is monitored over a 24-hour period. This embodiment optimizes the effect of ventilation frequency on 5-ALA synthesis. CO2 is introduced every 8h, 12h, or 24h, and the CO2 ventilation pressure is 5kgf / cm 2 , ventilation time is 5min.

[0062] Depend on Figure 8 It can be seen that the yield of 5-ALA electrocatalytically synthesized with CO2 as the only carbon source is increased, and when CO2 is passed once every 12 hours, the yield can reach 110 mg / L. The formate dehydrogenase present in Pichia pastoris reduces CO2 to formic acid, which is supplied to the methylotrophic Pichia pastoris for electrocatalytic synthesis of the target product.

[0063] Example 7 Optimization of the electron mediator methyl violet concentration

[0064] The optimized electrocatalytic synthesis system of 5-ALA was constructed according to Example 6, with the concentration of the electron mediator changed to 0.15 mM, 0.2 mM, 0.5 mM, and 0.8 mM.

[0065] The results are as follows Figure 9 As shown in the figure, it can be seen that at a concentration of 0.2 mM electron mediator, the 5-ALA production has a qualitative leap, reaching 220 mg / L.

[0066] Example 8 13 C tracer labeling

[0067] The electrocatalytic synthesis system of 5-ALA was constructed according to Example 7. 13 C-labeled CO2 is relatively expensive, so we only use the CO2 provided by Anhui Zesheng Company. 13 C-labeled NaHCO3, the initial addition concentration of NaHCO3 was 50 mM, and NaHCO3 was added once every 24 h during the electrochemical synthesis process, with each addition concentration of 50 mM. The electrochemical synthesis time was 72 h. 13 C-labeled NaHCO3 and 13 C-labeled CO2 is a different supply form, but its essence is to use CO2 as the carbon source for detection. 13 The amount of C-labeled 5-ALA product was detected by gas chromatography-mass spectrometry (GC-MS). 13 C labeled amount. 13The C-labeled amount detection method and the 5-ALA yield detection method use the same gas chromatography conditions. The difference is that a specific m / z ratio needs to be monitored to detect 5-ALA and its 13 The amount of C-labeled products, especially at m / z ratios of 159–164 and 214–219, corresponds to different amounts of 5-ALA molecules and their fragments after derivatization. 13 The mass of the C isotope.

[0068] The results are as follows Figure 10 As shown, 13 The C labeling reached 86%, while the theoretical labeling should be 92% (product increased from 5.1 mg / L to 74.7 mg / L). This demonstrates that the engineered Pichia pastoris can grow using CO2 as a carbon source, driven by a microbial electrochemical system, utilizing the reduced glycine pathway and the HEM1 enzyme.

[0069] In summary, the present invention discloses a method for electrosynthesizing 5-ALA using Pichia pastoris. A recombinant Pichia pastoris bioelectrochemical system is constructed. By changing the carbon source supply form, carbon source supply concentration, frequency, voltage, and electron mediator concentration, the electrocatalytic system of the recombinant Pichia pastoris is regulated to achieve a breakthrough in 5-ALA production.

[0070] The present invention provides a method and concept for electrosynthesizing 5-aminolevulinic acid in Pichia pastoris. Numerous methods and approaches exist for implementing this technical solution. The foregoing merely represents a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.

Claims

1. A method for electrosynthesizing 5-aminolevulinic acid by Pichia pastoris, characterized in that: An electrochemical device is used which is divided into a cathode chamber and an anode chamber by a proton exchange membrane. A catholyte is added to the cathode chamber and an anode chamber, and an external voltage is applied to the electrochemical device to electrochemically synthesize 5-aminolevulinic acid. Wherein, the cathode liquid comprises a carbon source, a Pichia pastoris resuspension solution and an electron mediator; The Pichia pastoris is a Pichia pastoris that produces 5-aminolevulinic acid.

2. The method according to claim 1, characterized in that The Pichia pastoris is Pichia pastoris PMORG09-HEM1; and the electron mediator is methyl viologen.

3. The method according to claim 1, characterized in that In the cathode liquid, the OD of Pichia pastoris 600 The concentration of the electron mediator is 0.15 to 0.8 mM.

4. The method according to claim 1, wherein The Pichia pastoris resuspension is prepared according to the following method: the Pichia pastoris strain is activated and inoculated into a YPD liquid culture medium supplemented with an inducer, and after induction culture for 48 to 120 hours, the strain is resuspended in an inorganic salt culture medium; wherein the inducer is added once every 12 to 24 hours during the induction culture process.

5. The method according to claim 4, characterized in that The inducer is methanol; the initial added concentration of the inducer is 0.8-1.2 g / L; the amount of the inducer added each time during the induction culture is 0.8-1.2 g / L based on the volume of the initial YPD liquid culture medium.

6. The method according to claim 1, characterized in that The carbon source is any one of formic acid, formate or CO2 or a combination of them.

7. The method according to claim 6, characterized in that When the carbon source is formic acid or formate, the initial concentration of the carbon source in the cathode liquid is 1 to 8 g / L. During the electrochemical synthesis process, the carbon source is added once every 12 to 24 hours, and the amount of the carbon source added each time is 2 to 3 g / L based on the volume of the initial cathode liquid. When the carbon source is CO2, CO2 is added to the cathode liquid in the form of NaHCO3 and / or CO2 gas, wherein the concentration of NaHCO3 in the cathode liquid is 10-80 mM; CO2 gas is aerated in the cathode liquid at an aeration pressure of 1-5 kgf / cm 2 The aeration time is 2 to 5 minutes. During the electrochemical synthesis process, CO2 gas is added once every 8 to 24 hours. The aeration pressure of each CO2 gas addition is 5 kgf / cm 2 , the aeration time is 5 minutes.

8. The method according to claim 1, characterized in that The formula of the anolyte includes: NaHPO4 2-3g / L, NaH2PO4 2-3g / L, NaCl 1-2g / L and dithiothreitol 0.001-0.002g / L.

9. The method according to claim 1, characterized in that The applied voltage provided to the electrochemical device is -0.8 to -1.2 V; The electrochemical synthesis was carried out at 30°C.

10. The method according to claim 1, characterized in that Graphite felt is used as an electrode in the cathode chamber, and platinum sheet is used as an electrode in the anode chamber.

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