Method for electrically synthesizing 5-aminolevulinic acid by using pichia pastoris

Abstract

The invention discloses a method for electrically synthesizing 5-aminolevulinic acid by using pichia pastoris. According to the method, based on construction of pichia pastoris for reducing a glycine pathway and a 5-ALA synthesis pathway, a microbial electrochemical system is used for providing external electric energy to realize electric synthesis of 5-ALA. According to the method, pichia pastoris is selected as a research object, an efficient carbon sequestration glycine reduction pathway and a 5-ALA generation pathway are constructed in the earlier stage, the recombinant pichia pastoris is applied to a microbial electrochemical system, formic acid or CO2 is used as a carbon source, electric energy supplied by the microbial electrochemical system is used as an energy source, and the microbial electrochemical system is constructed. And the generation condition of the pichia pastoris 5-ALA under the electric energy condition is researched.

Description

Technical Field

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

[0002] Excessive CO2 emissions have led to a continuous increase in the concentration of CO2 in the atmosphere, which in turn has caused a series of environmental problems such as the greenhouse effect and glacier melting. In order to mitigate the greenhouse effect, the development of CO2 fixation and utilization technology has become one of the current research focuses. Effective CO2 fixation and utilization methods can turn waste CO2 into "treasure", providing a possibility for alleviating the increase in atmospheric CO2 content and the ecological problems it causes.

[0003] Microbial CO2 fixation technology provides a promising new approach to achieve "carbon neutrality". Microorganisms can convert CO2 into useful compounds under environmentally friendly conditions through specific CO2 fixation pathways and energy input. In the process of microbial CO2 fixation, the selection of CO2 fixation pathways and energy supply are key. The Reductive Glycine Pathway (RGP) is a biochemical process used for CO2 fixation in microorganisms. This pathway is an effective means in CO2 fixation technology, especially in environmental and industrial applications that strive to achieve carbon neutrality goals. The Reductive Glycine Pathway has attracted the attention of researchers for its efficient carbon fixation capacity and low energy consumption.

[0004] 5-Aminolevulinic acid (5-ALA) is a key non-protein amino acid that is 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, especially in biosynthesis. Microbial synthesis, as an environmentally friendly 5-ALA synthesis scheme, has the potential, but is limited in commercial application by low yields. Among these biosynthetic methods, the use of yeast as a host for 5-ALA production is particularly faced with 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 has a weak natural porphyrin metabolic flux, which means that their ability to synthesize 5-ALA in an unmodified state is limited.

[0005] As a methylotrophic microorganism, Pichia pastoris has become an ideal host for studying CO2 fixation and metabolic reconstruction due to its metabolic flexibility and industrial adaptability. By constructing an efficient carbon fixation and reduction glycine pathway and a 5-ALA synthesis pathway, Pichia pastoris can achieve efficient CO2 fixation and synthesis of 5-ALA under the condition of external electrical energy. The design of this system provides new technical support for the realization of large-scale industrial 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 provide a method for electrosynthesizing 5-aminolevulinic acid (5-ALA) by Pichia pastoris in view of the deficiencies in the prior art. 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 scheme is as follows:

[0008] The present invention provides a method for electrosynthesizing 5-aminolevulinic acid by Pichia pastoris, using an electrochemical device divided into a cathode chamber and an anode chamber by a proton exchange membrane, adding a cathode liquid into the cathode chamber, adding an anode liquid into the anode chamber, and providing an external voltage to the electrochemical device to electrochemically synthesize 5-aminolevulinic acid (5-ALA). The cathode liquid includes a carbon source, a Pichia pastoris resuspension liquid, and an electron mediator; and the Pichia pastoris is a Pichia pastoris that produces 5-aminolevulinic acid.

[0009] Wherein, the electron mediator is methyl viologen; 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, and the detailed information thereof 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 to 50, and the concentration of the electron mediator is 0.15 to 0.8 mM. Preferably, the OD of Pichia pastoris is 600 The molecular weight of the electron mediator is 40, and 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 the following method: activating the Pichia pastoris strain and inoculating it into a YPD liquid medium with an inducer added, inducing and culturing for 48 to 120 hours, and then resuspending it in an inorganic salt medium, and adding the inducer once every 24 hours during the induction culture. Preferably, the formula of the inorganic salt medium is: (NH4)2SO42.5g / L, KH2PO414.4g / L and MgSO4·7H2O0.5g / L. The induction culture time is 72h.

[0012] Wherein, the inducer is methanol; the initial addition 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. 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, 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 5 min. 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 50 mM, and the aeration pressure of CO2 gas in the cathode liquid is 5 kgf / cm 2 The aeration time is 5 minutes. During the electrochemical synthesis process, CO2 gas is added once every 12 hours. The aeration pressure each time is 5 kgf / cm 2 , the aeration time is 5 minutes.

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

[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 an electrode in the cathode chamber, and platinum sheet is used as an electrode in the anode chamber.

[0019] Beneficial effects:

[0020] Compared with the prior art, the present invention provides a method for electrosynthesizing 5-aminolevulinic acid by 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 in the electrochemical device, thereby obtaining a strain with a better 5-ALA yield under the condition of using only formic acid or CO2 as the sole carbon source and external electrical energy as the energy source. The specific advantages are:

[0021] (1) The Pichia microbial electrochemical device constructed in the present invention can use electrical energy as an energy source to drive microbial metabolic activities and directly synthesize target products through electrical energy, which meets the requirements of carbon neutrality and green production. Compared with chemical or light energy supply, electrical energy can more accurately control the supply of electrons and the demand for reducing power, and is 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 in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[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 optimized for culture medium using formic acid as the carbon source.

[0027] Figure 4 This is the analysis diagram of 5-ALA production optimized by induction time with formic acid as 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 formic acid feed concentration optimization.

[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 when CO2 is the only carbon source.

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

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

[0034] The present invention is further illustrated below through the description of specific implementation modes, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not deviate from the basic idea 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, 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, NaCl1.45g / L, DTT 0.002g / L, pH is 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 of autotrophic Pichia pastoris. The specific construction method is detailed in the 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 detection method of 5-ALA production is as follows:

[0040] First, 5-ALA needs to be properly 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: collect 2-3 mL of bacterial solution after the culture is completed, centrifuge at 4°C and 8800g for 5 minutes, resuspend the bacteria with 1 mL of pre-cooled ddH2O, centrifuge at 4°C and 8800g for 5 minutes to wash the bacteria, repeat 2-3 times, add 200 μL of 6M HCl, mix the bacteria and incubate at 99°C for 24 hours to lyse cell proteins, and after the incubation, add an equal volume of 6M NaOH to neutralize the HCl, centrifuge at 4°C and 8800g for 1 minute, collect the supernatant and lyophilize. The lyophilized sample is in the form of hollow sheets, either white or light yellow, and is subjected to pre-column derivatization (derivation reagent is added according to the lyophilized volume, and the amount of bacteria needs to be reduced if the volume is too large. Standard samples are prepared by simultaneous derivatization of standard products). 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 supernatant is placed in an injection bottle for GC-MS detection.

[0041] High-resolution GC-MS (Thermo TRACE 1310 gas chromatograph, Orbitrap Exploris GC240 mass spectrometer, Thermo Scientific 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 set as follows: 60 °C for 2 min, then increased to 200 °C at a rate of 5 °C / min, and then increased to 320 °C at a rate of 10 °C / min and held for 3 min. The mass spectra of amino acids were performed in the mass range of 50–650 m / z with an acquisition rate of 5 spectra per second. The temperatures of the ion source and transfer line were 250 °C and 290 °C, respectively. Electron ionization was performed at 70 eV. The carrier gas was helium with a constant flow rate of 1 mL / min. The sample injection volume was 1 μL, and each sample was repeated three times. 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 shaking incubator, and methanol inducer was added at a cycle of 24 hours, and the concentration of methanol added each time was 1 g / L (based on the volume of the initial MM medium). After 120 hours of shaking 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 by a membrane (0.22 μm) in the form of a methyl viologen aqueous solution and then added to the resuspension, and the initial OD of the strain was 2.5 %. 600 40 portions were filled into the cathode chamber of the microbial electrochemical device, and 50 mL of the anolyte was poured into the anode chamber of the microbial electrochemical device. One group was subjected to voltage (-0.8 V) and the other group was not subjected to voltage. The 5-ALA production was monitored at a 24-hour cycle.

[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 the field. The constructed electrochemical device is soaked in 75% alcohol and left to stand overnight. The electrochemical device is taken out after soaking, placed in a clean bench, irradiated with ultraviolet light and blown dry. The structure of the electrochemical device is shown in the figure below. Figure 1 shown.

[0045] Electric energy drive output analysis chart 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 to reduce glycine 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] According to the method described in Example 1, an electric energy driven glycine reduction system was constructed to synthesize 5-ALA. The MM medium in the induction period described in Example 1 was replaced with YPD medium. The other conditions remained unchanged and the 5-ALA yield was monitored at a cycle of 24 hours. The results showed that after the MM medium was replaced with YPD medium, the yield increased from 20 mg / L to 50.9 mg / L, which was twice as much as before. 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 4 shown.

[0051] Depend on Figure 4 It can be seen that the 5-ALA production was not much different when the induction time was 72h, 96h and 120h, while the 5-ALA production was lower when the induction time was 48h. Considering the time and economic cost, the subsequent experiments chose to drive 5-ALA synthesis by whole-cell electricity in high-density cells after 72h 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 an appropriate induction time was constructed to synthesize 5-ALA. The formic acid concentration was changed to 3 g / L, 5 g / L, and 8 g / L, and the other conditions remained unchanged. The 5-ALA production was monitored over a 24-hour period. The results are shown in Figure 5 shown.

[0054] from Figure 5It can be seen that the higher the formic acid concentration, the stronger the inhibitory effect. The formic acid concentration of 3g / L is just enough for Pichia pastoris to use for catalysis, the formic acid consumption can reach nearly 100%, and the 5-ALA production can reach 51mg / L. Therefore, it can be concluded that the formic acid concentration of 3g / L is most suitable for Pichia pastoris to electrocatalyze the synthesis of 5-ALA.

[0055] Since the 5-ALA production was still very low and the formic acid was almost completely utilized, we considered whether the formic acid supply was insufficient. Therefore, we chose to add 2 g / L formic acid feed (based on the initial cathode liquid 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] According to the method of Example 4, a suitable voltage electric driven reduction system of glycine was constructed to synthesize 5-ALA, and the applied voltages were -0.8 V, -1.2 V and -1.5 V. The other conditions remained unchanged. The 5-ALA production was monitored in a 24-hour cycle.

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

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

[0061] Pick the positive clone of strain PMORG09-HEM1 into 10 mL YPD liquid medium and culture it overnight at 30°C and 200 rpm until 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 at a cycle of 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 shaking 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 of the strain was 2. 60040 parts 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 ventilation tube equipped with a 0.2 μm microporous filter membrane was connected to a CO2 gas cylinder (the ventilation pressure of the CO2 was 5 kgf / cm 2 , ventilation time is 5min), and cathode liquid is introduced into the other end. Voltage is applied -0.8V. 5-ALA production is monitored in a 24-hour cycle. This embodiment optimizes the effect of ventilation frequency on the synthesis of 5-ALA, CO2 is passed once every 8h, 12h or 24h, and the ventilation pressure of CO2 each time 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] According to the optimized electrocatalytic synthesis system of 5-ALA constructed in Example 6, the concentration of the electron mediator was changed to 0.15 mM, 0.2 mM, 0.5 mM, and 0.8 mM.

[0065] The results are as follows Fig. 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 label

[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, each addition concentration was 50 mM, and the electrochemical synthesis time was 72 h. 13 C-labeled NaHCO3 13 C-labeled CO2 is a different supply form, and 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 GC 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 Fig.10 As shown, 13 The C labeling amount reached 86%, and the theoretical labeling amount should be 92% (the product increased from 5.1 mg / L to 74.7 mg / L). This shows that the engineered Pichia pastoris can grow with CO2 as a carbon source under the action of the reduced glycine pathway and the HEM1 enzyme, driven by the microbial electrochemical system.

[0069] In summary, the present invention discloses a method for electrosynthesizing 5-ALA by Pichia pastoris, constructs a recombinant Pichia pastoris bioelectrochemical system, and regulates the electrocatalytic system of the recombinant Pichia pastoris by changing the carbon source supply form, carbon source supply concentration and frequency, voltage, and electron mediator concentration, thereby achieving a breakthrough in 5-ALA production.

[0070] The present invention provides a method and idea for electrosynthesizing 5-aminolevulinic acid in Pichia pastoris. There are many methods and approaches to realize the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be considered as the protection scope of the present invention. All components not specified in this embodiment can be realized by 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 cathode liquid is added to the cathode chamber, an anode liquid is added to the anode chamber, and an external voltage is provided to the electrochemical device to electrochemically synthesize 5-aminolevulinic acid; Wherein, the cathode liquid comprises a carbon source, a Pichia pastoris resuspension liquid 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 solution, the OD of Pichia pastoris 600 is 30-50, and the concentration of the electron mediator is 0.15-0.8 mM.

4. The method according to claim 1, characterized in that 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 with an inducer added thereto, 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 addition 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 initial YPD liquid culture medium volume.

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 initial cathode liquid volume. 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 2-3 g / L, NaH2PO4 2-3 g / L, NaCl 1-2 g / L and dithiothreitol 0.001-0.002 g / 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.

Images