Method for recombinant pichia pastoris growth on formic acid or co2 based on electrochemical devices

Abstract

The application discloses a method for growth of recombinant Pichia pastoris based on an electrochemical device by using formic acid or CO2. The method uses formic acid or CO2 as a carbon source, and uses a microbial electrochemical system to provide external electric energy to realize application of the recombinant Pichia pastoris to growth by using electric energy. In the application, a natural methylotrophic strain Pichia pastoris is selected as a research object, and in an early stage, the Pichia pastoris with high carbon fixation efficiency by overexpressing a reductive glycine pathway is constructed. The recombinant Pichia pastoris is applied to the microbial electrochemical system, formic acid or CO2 is used as the carbon source, and electric energy supplied by the microbial electrochemical system is used as an energy source, so that the effect of the electric energy on growth and metabolism of the Pichia pastoris is explored.

Description

Technical Field

[0001] This invention belongs to the field of microbial electrochemistry, specifically relating to a method for the growth of recombinant Pichia pastoris using formic acid or CO2 based on an electrochemical device. Background Technology

[0002] Energy demand is a key factor affecting CO2 fixation efficiency in the CO2 fixation pathway. The carbon in CO2 is in the +4 oxidation state, and converting it into biomass or chemicals requires a large amount of energy and reducing power. Commonly used chemical energy sources such as glucose, acetic acid, methanol, and formic acid can provide the energy needed for carbon fixation. Formate, being a product of the reduction of carbon dioxide with a single pair of electrons, is one of the simplest organic compounds, providing cells with carbon and reducing power. The reduced glycine pathway is a metabolic pathway of formate assimilation, which can utilize formate and CO2 for biosynthesis. In the reduced glycine pathway, the production of pyruvate from formate and CO2 requires 3 molecules of NADPH and 2 molecules of ATP. However, using chemical energy leads to a significant loss of carbon atoms through catabolism, resulting in low economic efficiency.

[0003] Microbial electrochemical systems are an emerging technology that combines microorganisms with electrochemistry. The basic principle of microbial electrosynthesis is to use an electrochemical system to enable microorganisms to convert exogenous electrons into usable reducing power within the cell. Therefore, microbial electrochemical systems can provide energy for microbial carbon dioxide reduction and cell growth and maintenance. Using electricity to drive biological carbon fixation processes can improve the efficiency of biological carbon fixation and carbon atom economy, while also solving the difficulties of electrical energy storage and utilization.

[0004] CO2 is the most abundant greenhouse gas on Earth, but it is also the most abundant carbon resource in nature. Therefore, developing microbial electrochemical systems for CO2 fixation and utilization, using CO2 as a carbon feedstock, and realizing CO2-based biomanufacturing can not only alleviate environmental problems such as the greenhouse effect, but also promote the sustainable development of the biotechnology manufacturing industry. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for the growth of recombinant Pichia pastoris using formic acid or CO2 based on an electrochemical device. This method uses formic acid or CO2 as a carbon source and utilizes a microbial electrochemical device to realize the application of electrical energy by recombinant Pichia pastoris.

[0006] A method for growing recombinant Pichia pastoris using formic acid or CO2 based on an electrochemical device includes the following steps:

[0007] Step 1, Construct recombinant Pichia pastoris;

[0008] Step 2: Select the anode, cathode, and reference electrode to build the electrochemical device;

[0009] Step 3: Select positive clones of recombinant Pichia pastoris, activate them overnight in a shaker at 30°C, inoculate the activated strain into M9 medium, add an electron mediator, stir well, transfer to an electrochemical device as a cathode liquid, add a carbon source, set the electrochemical reaction conditions, apply an external voltage of (-0.8V) - (-1.5V), and culture at 30°C.

[0010] As an improvement, when the recombinant Pichia pastoris mentioned in step 1 is GS115-rGly-ΔDAS-ΔFDH, GS115-ΔFDH or GS115-rGly-ΔFDH, the carbon source in step 3 is formic acid.

[0011] As an improvement, when the recombinant Pichia pastoris described in step 1 is GS115-rGly-ΔDAS, the carbon source in step 3 is CO2.

[0012] As an improvement, the anode (counter electrode) in step 2 is made of platinum sheet, the cathode (working electrode) is made of indium metal or graphite felt, and the reference electrode is an AgCl / Ag electrode.

[0013] As an improvement, in step 3, the electron mediator is neutral red, methyl violane, emodin, or potassium ferricyanide, with a concentration of 0.1 mM-0.5 mM.

[0014] Beneficial effects:

[0015] Compared with existing technologies, this invention utilizes an electrochemical device to grow recombinant Pichia pastoris using formic acid or CO2, constructing a recombinant Pichia pastoris bioelectrochemical system. By optimizing the types and concentrations of electron mediators and electrochemical parameters within the electrochemical device, the growth environment of the recombinant Pichia pastoris is controlled, resulting in strains with superior growth under conditions where formic acid or CO2 is the sole carbon source and external electrical energy is the energy source. This invention also represents a breakthrough in growth metabolism during the cultivation process, pioneering CO2-based biomanufacturing technology.

[0016] The specific advantages are:

[0017] 1. The recombinant Pichia pastoris microbial electrochemical device constructed in this invention can provide reducing power to cells, enabling recombinant Pichia pastoris with the formic acid dissimilatory pathway knocked out to grow with formic acid as the sole carbon source and electrical energy as the energy source, thereby reducing carbon source loss and improving carbon source utilization efficiency.

[0018] 2. In this invention, a microbial electrochemical device is used in conjunction with formic acid dehydrogenase to electrocatalyze the reduction of CO2 to formic acid, thereby eliminating the need for formic acid as a carbon source and providing energy and reducing power for recombinant Pichia pastoris, thus achieving CO2 as the sole carbon source. Attached Figure Description

[0019] Figure 1 This is a structural diagram of a microbial electrochemical device;

[0020] Figure 2 A graph showing the growth characteristics of a recombinant Pichia pastoris strain without formate dehydrogenase knockout;

[0021] Figure 3 Growth characteristics analysis of recombinant Pichia pastoris strain with formate dehydrogenase knocked out;

[0022] Figure 4 CV scan of the electrochemical device used to inoculate the recombinant strain;

[0023] Figure 5 CV scan of the chemical apparatus without the addition of recombinant strains;

[0024] Figure 6 This is a growth curve of the recombinant strain using formic acid as a carbon source.

[0025] Figure 7 This is a growth curve of the recombinant strain when CO2 is used as the carbon source;

[0026] Figure 8 To investigate the effect of metal electrodes on the growth curves of recombinant strains. Detailed Implementation

[0027] The present invention will be further described below through specific embodiments, 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 depart from the basic idea of ​​the present invention, they are all within the scope of the present invention.

[0028] Example 1: Analysis of the growth characteristics of recombinant Pichia pastoris

[0029] Positive clones of strains 1 (GS115-ΔFDH), 3 (GS115-ΔFDH-rGly), 4 (GS115-rGly), 5 (GS115-ΔFDH-rGly-ΔDAS), and 6 (GS115-rGly-ΔDAS) were picked and added to 10 mL of YPD medium and cultured overnight at 30°C and 200 rpm until OD. 600 Reaching 2-3. The activated strain was then subjected to initial OD... 6000.1 was transferred to M9 medium and incubated on a shaker at 30°C. OD was monitored every 24 hours. 600 .

[0030] The above YPD medium (g / L) consists of: peptone 20g, yeast extract 10g, glucose 20g, and pure water as the solvent.

[0031] M9 medium (g / L): Na2PO4·7H2O 12.8, KH2PO4 3.0, NaCl 0.5, NH4Cl 1, MgSO4·7H2O 0.492, CaCl2·6H2O 0.02191, solvent is pure water.

[0032] The growth characteristics of different recombinant Pichia pastoris strains are shown in the figure below. Figure 2 , 3 As shown. From Figure 2 , 3 As can be seen, strains that knock out the formic acid dissimilatory pathway suffer from energy deficiency and cannot grow using formic acid as their sole carbon source.

[0033] Example 2: Construction of a microbial electrochemical device

[0034] according to Figure 1 The electrochemical device shown is constructed with a proton exchange membrane separating the cathode and anode chambers. The anode (counter electrode) is made of platinum sheet, the cathode (working electrode) is made of graphite felt, and the reference electrode is an AgCl / Ag electrode. All other components and connections are standard practices in the field. The constructed electrochemical device was immersed in 75% ethanol and left to stand overnight. Simultaneously, serum bottles containing pure water and Erlenmeyer flasks equipped with M9 culture medium were sterilized at 121°C in an autoclave. After sterilization, the devices were dried in an oven until ready for use. The immersed electrochemical device was then removed, placed in a clean bench, and dried under ultraviolet light.

[0035] Positive clones of strain 1: GS115‐ΔFDH were picked and cultured overnight at 30℃ and 200 rpm in 10 mL YPD medium until their OD values ​​reached 100%. 600 Reaching 2-3. The activated strain was then... 600 0.1 Transfer to M9 medium, add methyl viologen to the M9 basal medium via membrane. Aliquot into the cathode chamber of the microbial electrochemical device. Pour 50 mL of anolyte into the anode chamber of the microbial electrochemical device. The anolyte composition is NaHPO4·12H2O 2.51 g / L, NaH2PO4·2H2O 2.81 g / L, NaCl 1.45 g / L, 0.02% DTT, pH 7.0.

[0036] The microbial electrochemical device was tested using cyclic voltammetry, and the results are as follows: Figure 4 , Figure 5 As shown in the CV scan results, obvious redox peaks appeared in all experimental groups, and the peak potentials during the oxidation process were all between -0.5 V and -0.6 V. This indicates that a large number of electroactive microorganisms were attached to the cathode surface within this potential range. It also proves that when methyl viologen (MV) is used as an electron mediator, the electroactive microorganisms can be used to transfer electrons normally and exert their effect.

[0037] Example 3: Investigation of the growth of recombinant Pichia pastoris using formic acid in a microbial apparatus

[0038] Following the method in Example 3, a microbial electrochemical device was constructed, with the addition of 0.5 mM methyl viologen; all other steps remained unchanged. OD was monitored over a 24-hour period. 600 The result is as follows Figure 7 As shown.

[0039] Depend on Figure 6 It can be seen that recombinant Pichia pastoris can utilize formic acid for growth under a microbial electrochemical system.

[0040] Example 4: Investigation of the growth of recombinant Pichia pastoris using CO2 in a microbial apparatus

[0041] A microbial electrochemical device was constructed according to Example 3, with the strain changed to GS115-rGly-ΔDAS, the electron mediator being 0.5 mM methyl viologen, and the carbon source changed to 10 mM NaHCO3. OD was monitored on a 24-hour cycle. 600 Each time a sample is taken, CO2 is passed through for five minutes.

[0042] The results are as follows Figure 7 As shown in the figure, under the action of the microbial electrochemical device, the recombinant Pichia pastoris GS115‐rGly‐ΔDAS can grow with CO2 as the sole carbon source, while the recombinant yeast with FDH knocked out cannot grow.

[0043] Example 5: Effects of different metal electrodes on the CO2-utilized growth of recombinant Pichia pastoris

[0044] A microbial electrochemical device was constructed according to Example 5, with the working electrode changed to indium (In).

[0045] The results are as follows Figure 8 As shown in the figure, CO2 is electrocatalyzed to produce formic acid using indium as an electrode. The recombinant strain GS115-rGly-ΔDAS-ΔFDH assimilates formic acid for cell growth. Using carbon felt as an electrode, formic acid dehydrogenase catalyzes the reduction of CO2, and GS115-rGly-ΔDAS can grow using CO2 as a carbon source.

[0046] In summary, this invention constructs a recombinant Pichia pastoris bioelectrochemical system based on an electrochemical device using formic acid or CO2 for growth. By optimizing the types and concentrations of electron mediators and electrochemical parameters within the electrochemical device, the growth environment of the recombinant Pichia pastoris is controlled, resulting in strains with superior growth under conditions where formic acid or CO2 is the sole carbon source, and breakthroughs in growth metabolism during the cultivation process.

Claims

1. A method for growing recombinant Pichia pastoris based on an electrochemical device using formic acid or CO2, characterized in that, The specific steps are as follows: Step 1, constructing recombinant Pichia pastoris, wherein the recombinant Pichia pastoris is GS115-rGly-ΔDAS-ΔFDH, GS115-ΔFDH, GS115-rGly-ΔFDH or GS115-rGly-ΔDAS; Step 2, selecting an anode, a cathode and a reference electrode to build an electrochemical device; Step 3, selecting a positive clone of the recombinant Pichia pastoris, activating the culture overnight on a shaking table, inoculating the activated strain into a M9 medium, adding methyl viologen, stirring uniformly, transferring into the electrochemical device as a cathode liquid, adding a carbon source, setting the conditions of the electrochemical reaction, applying an external voltage (-0.8V)-(-1.5V), and culturing at 30°C, thereby obtaining the product; when the recombinant Pichia pastoris in step 1 is GS115-rGly-ΔDAS-ΔFDH, GS115-ΔFDH or GS115-rGly-ΔFDH, the carbon source in step 3 is formic acid; when the recombinant Pichia pastoris in step 1 is GS115-rGly-ΔDAS, the carbon source in step 3 is CO2; the material of the anode in step 2 is platinum, the material of the cathode is metal indium or graphite felt, and the reference electrode is an AgCl / Ag electrode.

2. The method for growing Pichia pastoris using formic acid or CO2 based on an electrochemical device according to claim 1, characterized by, the concentration of methyl viologen in step 3 is 0.1-0.5mM.

Images