CN116103165A - Recombinant pichia pastoris utilizing formic acid or CO based electrochemical devices 2 Method of growth - Google Patents
Recombinant pichia pastoris utilizing formic acid or CO based electrochemical devices 2 Method of growth Download PDFInfo
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- CN116103165A CN116103165A CN202211697744.4A CN202211697744A CN116103165A CN 116103165 A CN116103165 A CN 116103165A CN 202211697744 A CN202211697744 A CN 202211697744A CN 116103165 A CN116103165 A CN 116103165A
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- 241000235058 Komagataella pastoris Species 0.000 title claims abstract description 44
- 230000012010 growth Effects 0.000 title claims abstract description 32
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- 235000019253 formic acid Nutrition 0.000 title claims abstract description 24
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Abstract
The invention discloses an electrochemical device-based recombinant Pichia pastoris utilizing formic acid or CO 2 A method of growing. The method uses formic acid or CO 2 The application of the recombinant pichia pastoris in electric energy growth is realized by using the microbial electrochemical system to provide external electric energy for a carbon source. The invention selects natural methyl nutrition strain Pichia pastoris as a research object, constructs the Pichia pastoris with high-efficiency carbon fixation through over-expression reduction glycine pathway in the early stage, applies the recombinant Pichia pastoris to a microbial electrochemical system, and uses formic acid or CO 2 As a carbon source, the electric energy supplied by a microbial electrochemical system is used as an energy source, and the action condition of the electric energy on the growth and metabolism of pichia pastoris is explored.
Description
Technical Field
The invention belongs to the field of microbial electrochemistry, and in particular relates to an electrochemical device-based recombinant pichia pastoris utilizing formic acid or CO 2 A method of growing.
Background
The energy requirement is CO 2 Influence of CO in the fixation pathway 2 The key factor in the efficiency of fixation. CO 2 The carbon in (a) is in the +4 oxidation state and requires a great deal of energy and reducing power in the process of converting it into biomass or chemicals. Chemical energy such as glucose, acetic acid, methanol, formic acid, etc. is commonly used to provide the energy required for carbon fixation, wherein formate is a product of reduction of carbon dioxide by single pair electrons, which becomes one of the simplest organic compounds, and can provide carbon and reducing power for cells. The reduced glycine pathway is a metabolic pathway for formate assimilation that can utilize formate and CO 2 Biosynthesis is carried out. In the reductive glycine pathway, formate and CO 2 3 molecules of NADPH and 2 molecules of ATP are required for the substrate to produce pyruvate. However, the chemical energy is used for providing energy, so that a large amount of carbon passes through a dissimilation pathway to cause the loss of carbon atoms, and the economic efficiency is low.
Microbial electrochemical systems are an emerging technology combining microorganisms with electrochemistry, wherein the basic principle of microbial electrosynthesis is to utilize an electrochemical system to convert exogenous electrons into intracellular available reducing power, so that the microbial electrochemical system can provide energy for reducing carbon dioxide and cell growth and maintenance. The electric energy is utilized to drive the biological carbon fixing process, so that the biological carbon fixing efficiency and the carbon atom economy can be improved, and the difficulty in electric energy storage and utilization can be solved.
CO 2 Is the most dominant greenhouse gas on the earth, but is also the most abundant carbon-resource in nature. Thus, in CO 2 Development of CO for carbon feedstock 2 Is realized based on CO 2 The biological manufacturing of the method not only can relieve environmental problems such as greenhouse effect and the like, but also is beneficial to sustainable development of biotechnology manufacturing industry.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an electrochemical device-based recombinant pichia pastoris utilizing formic acid or CO 2 Growth process with formic acid or CO 2 The application of the recombinant pichia pastoris to electric energy is realized by using a microbial electrochemical device as a carbon source.
Recombinant pichia pastoris utilizing formic acid or CO based on electrochemical device 2 A method of growing comprising the steps of:
step 2, selecting an anode, a cathode and a reference electrode, and constructing an electrochemical device;
and 3, selecting positive clones of recombinant pichia pastoris, performing shaking table overnight activation culture at 30 ℃, taking the activated strains, inoculating the strains into a medium filled with M9, adding an electron mediator, stirring uniformly, transferring into an electrochemical device as a cathode liquid, adding a carbon source, setting the conditions of electrochemical reaction, applying voltage (-0.8V) - (-1.5V), and culturing at 30 ℃.
As an improvement, when the recombinant Pichia pastoris in the step 1 is GS115-rGly- ΔDAS- ΔFDH, GS115- ΔFDH or GS115-rGly- ΔFDH, the carbon source in the step 3 is formic acid.
As an improvement, when the recombinant Pichia pastoris in the step 1 is GS 115-rGly-delta DAS, the carbon source in the step 3 is CO 2 。
As an improvement, the anode (counter electrode) in the step 2 is made of a platinum sheet, the cathode (working electrode) is made of metal indium or graphite felt, and the reference electrode is an AgCl/Ag electrode.
As an improvement, the electron mediator in the step 3 is neutral red, methyl viologen, emodin or potassium ferricyanide, and the concentration is 0.1-mM-0.5-mM.
The beneficial effects are that:
compared with the prior art, the recombinant pichia pastoris based on the electrochemical device utilizes formic acid or CO 2 Growth method, construction of recombinant Pichia pastoris bioelectrochemical system, and optimization of electron mediator type and electron in electrochemical deviceThe mode of medium concentration and electrochemical parameters regulates and controls the growth environment of recombinant pichia pastoris, and the method obtains the recombinant pichia pastoris by only using formic acid or CO 2 As the sole carbon source, under the condition that external electric energy is an energy source, the strain with better growth state and breakthrough in growth metabolism in the culture process develop the method based on CO 2 Is a biological manufacturing technique of (a).
The method has the specific advantages that:
1. the recombinant pichia pastoris microbial electrochemical device constructed in the invention can provide reducing force for cells, so that the recombinant pichia pastoris knocked out of a formic acid catabolism pathway can grow by taking formic acid as a sole carbon source and electric energy as an energy source, thereby reducing the loss of the carbon source and improving the utilization efficiency of the carbon source;
2. in the invention, the microorganism electrochemical device is utilized to combine with formate dehydrogenase to electrically catalyze CO 2 Reducing to generate formic acid, eliminating the supply requirement of formic acid as carbon source, providing energy and reducing power for recombinant Pichia pastoris, realizing CO 2 Is the only carbon source.
Drawings
FIG. 1 is a block diagram of a microbial electrochemical device;
FIG. 2 is a graph showing growth characteristics of a non-knocked-out formate dehydrogenase recombinant Pichia pastoris strain;
FIG. 3 is a growth characterization of a knockout formate dehydrogenase recombinant Pichia pastoris strain;
FIG. 4 is a graph of growth of recombinant strain with neutral red as electron mediator;
FIG. 5 is a potential tower of the redox potential composition of different species at the cathode;
FIG. 6 is a CV scan of an electrochemical device inoculated with a recombinant strain;
FIG. 7 is a CV scan of a chemical device without added recombinant strain;
FIG. 8 is a graph showing the growth of recombinant strains when formic acid is used as a carbon source;
FIG. 9 is a graph of CO 2 A graph of the growth of the recombinant strain when it is the carbon source;
FIG. 10 is a graph of the growth of recombinant strains exploring a metal electrode pair.
Detailed description of the preferred embodiments
The invention is further illustrated by the following description of specific embodiments, which are not intended to be limiting, and various modifications or improvements can be made by those skilled in the art in light of the basic idea of the invention, but are within the scope of the invention without departing from the basic idea of the invention.
EXAMPLE 1 analysis of growth Properties of recombinant Pichia pastoris
Respectively picking strains 1: GS 115-. DELTA.FDH, strain 2: GS115, strain 3: GS115- ΔFDH-rGly, strain 4: GS115-rGly, strain 5: GS115- ΔFDH-rGly- ΔDAS, strain 6: positive cloning of GS 115-rGly-DeltaDAS (construction methods of Strain 1, strain 2, strain 3, strain 4, strain 5, strain 6, refer to CN 114634946A) to 10 mL YPD Medium, and culturing overnight at 30deg.C under 200rmp conditions to OD 600 Reaching 2-3. The activated strain was set at the initial OD 600 0.1 is transferred to M9 culture medium, put into 30 ℃ shaking table for culture, and OD is monitored at 24 hours as period 600 。
YPD Medium (g/L) above: 20 parts of peptone, 10 parts of yeast powder and 20 parts of glucose, wherein the solvent is pure water
M9 medium (g/L): na (Na) 2 PO 4 ·7H 2 O 12.8、KH 2 PO 4 3.0、NaCl 0.5、NH 4 Cl 1、MgSO 4 ·7H 2 O 0.492、CaCl 2 ·6H 2 O0.02191, the solvent is pure water.
The growth characteristics of different recombinant Pichia strains are shown in figures 2 and 3. As can be seen from fig. 2 and 3, the strain knocked out the formate catabolism pathway causes energy deficiency and cannot grow on formate as the sole carbon source.
Example 2 screening of electron mediators
Selecting strain 1: positive cloning of GS 115-. DELTA.FDH into 10 mL YPD culture was based on overnight culture at 30℃under 200rmp to its OD 600 And when the temperature reaches 2-3. The activated strain was set at the initial OD 600 0.1 was transferred to M9 medium and neutral red was added to M9 basal medium at a final concentration of 1mM by passing through the membrane. Packaging into sterilized serum bottle and microorganism electrochemical device, culturing at 30deg.C, and monitoring OD 600 . The results are shown in FIG. 4.
As seen in fig. 4: the OD value of the bacterial liquid 1-NR added with neutral red is higher than that of the bacterial liquid 1 without neutral red, the growth trend of the bacterial liquid 1-NR added with neutral red is steeper, the growth trend of the bacterial liquid 1-NR added with neutral red is mild, and the end OD is not obvious relative to the initial OD growth. On the other hand, the M9 medium added with neutral red alone without bacterial liquid was used as a blank and OD was measured, and it was found that OD also had a higher tendency to increase over a period of time. Meanwhile, part of flocculent precipitate is found in the bacterial liquid added with neutral red, so that the OD value of the bacterial liquid 1-NR is higher than that of the bacterial liquid 1.
Thus, by adjusting the electron mediator composition, it is compared whether or not a precipitate is generated therein, thereby excluding components that may generate a precipitate, so as to exclude the influence of the precipitate on the result.
The method comprises the following specific steps: different electron mediators: methyl viologen at a final concentration of 0.1mM, potassium ferricyanide at a final concentration of 0.1mM, emodin at a final concentration of 0.1mM, and neutral red at a final concentration of several concentration gradients (1 mM, 0.5mM, 0.1mM, 0.05 mM, 0.01 mM). It was observed whether or not precipitation was generated.
The results are shown in Table 1. As shown in Table 1, when the final concentration of NR was 0.5mM, flocculent precipitate was observed in the bacterial liquids of Nos. 1, 2, 3, 5, 6 and 7, and when the final concentration of NR was reduced to 0.1mM, no precipitate was observed, it was assumed that a certain amount of Neutral Red (NR) caused precipitation in the bacterial liquid, and thus the bacterial liquid OD was affected.
As can be seen from the potential tower consisting of the redox potentials of different substances at the cathode in FIG. 5, the redox potential of methyl viologen is located at the lower position, and is more easily utilized by an electron transfer chain, so that the effect of more effectively transferring electrons is achieved, and therefore, methyl viologen is selected as an electron mediator.
EXAMPLE 3 construction of microbial electrochemical device
According to the construction of the electrochemical device shown in fig. 1, a cathode chamber and an anode chamber are separated by a proton exchange membrane, the anode (counter electrode) is made of a platinum sheet, the cathode (working electrode) is made of graphite felt, a reference electrode is an AgCl/Ag electrode, and the other parts and the connection mode are all conventional means in the field. The built electrochemical device is placed in 75% alcohol for soaking and standing overnight, meanwhile, a serum bottle filled with pure water and a conical bottle provided with an M9 culture medium are placed in a high-temperature sterilization pot for sterilization at 121 ℃, and after sterilization, the serum bottle and the conical bottle are placed in an oven for drying for standby. And taking out the soaked electrochemical device, putting the electrochemical device into an ultra-clean bench, and irradiating and drying by using an ultraviolet lamp.
Selecting strain 1: positive cloning of GS 115-. DELTA.FDH into 10 mL YPD culture was based on overnight culture at 30℃under 200rmp to its OD 600 Reaching 2-3. The activated strain is used as OD 600 0.1 transfer to M9 medium, adding methyl viologen to M9 basal medium via membrane. And dispensing the mixture into a cathode chamber of the microbial electrochemical device. 50mL of anolyte is poured into an anode chamber of the microbial electrochemical device, and the anolyte is formed by NaHPO 4 ·12H 2 O 2.51g/L、NaH 2 PO 4 ·2H 2 O2.81 g/L, naCl 1.45.45 g/L, 0.02% DTT, pH 7.0.
The results of the cyclic voltammetry test on the microbial electrochemical device are shown in fig. 6 and 7, and the CV scan results show that obvious oxidation-reduction peaks appear in the experimental group, and the peak potential in the oxidation process is between-0.5V and-0.6V, which shows that a large amount of electroactive microorganisms are attached to the surface of the cathode in the potential interval, and also proves that when the electrochemical device takes Methyl Viologen (MV) as an electron mediator, electrons can be normally transmitted by using the electroactive microorganisms, so that the effect of the electrochemical device is exerted.
Example 4 exploration of recombinant Pichia pastoris growth Using formate under microbial apparatus
A microbial electrochemical device was constructed according to the method of example 3, 0.5. 0.5mM methyl viologen was added, and the remaining steps were unchanged. OD monitoring at 24 hour period 600 . The results are shown in FIG. 7.
As can be seen from fig. 8, recombinant pichia pastoris can utilize formate growth under microbial electrochemical systems.
Example 5 exploration of recombinant Pichia pastoris utilization with CO under microbial devices 2 Growth
Construction of microbial electrochemical device according to example 3Changing the strain into GS 115-rGly-delta DAS, changing the electron mediator into 0.5mM methyl viologen, and changing the carbon source into 10mM NaHCO 3 . OD monitoring at 24 hour period 600 CO is introduced during each sampling 2 Five minutes.
As shown in FIG. 9, it can be seen that under the action of the microbial electrochemical device, the recombinant Pichia pastoris GS 115-rGly-DeltaDAS can be treated with CO 2 The only carbon source was grown, while FDH knocked out recombinant yeast was unable to grow.
EXAMPLE 6 use of different Metal electrodes on recombinant Pichia pastoris with CO 2 Effect of growth
A microbial electrochemical device was constructed as In example 5, with the working electrode changed to metallic indium (In).
As a result, as shown in FIG. 10, it can be seen that the electrocatalytic reduction of CO using indium metal as an electrode 2 Generating formic acid, assimilating formic acid by recombinant strain GS 115-delta FDH-rGly-delta DAS for cell growth, and catalyzing CO by formate dehydrogenase with carbon felt as electrode 2 Reduction of GS 115-rGly-DeltaDAS with CO 2 Is grown as a carbon source.
In summary, the electrochemical device-based recombinant Pichia pastoris of the present invention utilizes formic acid or CO 2 The growth method comprises the steps of constructing a recombinant pichia pastoris bioelectrochemical system, regulating and controlling the growth environment of the recombinant pichia pastoris by adopting a mode of optimizing the type of an electron mediator, the concentration of the electron mediator and electrochemical parameters in an electrochemical device, and obtaining the recombinant pichia pastoris by only using formic acid or CO 2 Under the condition of being the only carbon source, the strain with better growth state and breakthrough in growth metabolism in the culture process.
Claims (5)
1. Recombinant pichia pastoris utilizing formic acid or CO based electrochemical devices 2 The growth method is characterized by comprising the following specific steps:
step 1, constructing recombinant pichia pastoris;
step 2, selecting an anode, a cathode and a reference electrode, and constructing an electrochemical device;
and 3, selecting positive clones of recombinant pichia pastoris, performing activation culture on the clones overnight by a shaking table, inoculating the activated strains into a culture medium filled with M9, adding an electron mediator, uniformly stirring, transferring into an electrochemical device as a cathode solution, adding a carbon source, setting the conditions of electrochemical reaction, applying voltage (-0.8V) - (-1.5V), and culturing at 30 ℃.
2. The electrochemical-device-based recombinant pichia pastoris of claim 1, utilizing formate or CO 2 A method of growing, wherein when the recombinant pichia pastoris in step 1 is GS 115-rgy- Δdas- Δfdh, GS115- Δfdh or GS 115-rgy- Δfdh, the carbon source in step 3 is formic acid.
3. The electrochemical-device-based recombinant pichia pastoris of claim 1, utilizing formate or CO 2 The growth method is characterized in that when the recombinant Pichia pastoris in the step 1 is GS 115-rGly-delta DAS, the carbon source in the step 3 is CO 2 。
4. The electrochemical-device-based recombinant pichia pastoris of claim 1, utilizing formate or CO 2 The growth method is characterized in that in the step 2, the anode is made of a platinum sheet, the cathode is made of metal indium or graphite felt, and the reference electrode is an AgCl/Ag electrode.
5. The electrochemical-device-based recombinant pichia pastoris of claim 1, utilizing formate or CO 2 The method for growth is characterized in that the electron mediator in the step 3 is neutral red, methyl viologen, emodin or potassium ferricyanide, and the concentration is 0.1-0.5mM.
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| CN117106675A (en) * | 2023-10-23 | 2023-11-24 | 中国科学院天津工业生物技术研究所 | Electrochemical-microorganism integrated fixed CO 2 System, method and use for synthesizing single cell proteins |
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| CN117106675A (en) * | 2023-10-23 | 2023-11-24 | 中国科学院天津工业生物技术研究所 | Electrochemical-microorganism integrated fixed CO 2 System, method and use for synthesizing single cell proteins |
| CN117106675B (en) * | 2023-10-23 | 2024-01-05 | 中国科学院天津工业生物技术研究所 | Electrochemical-microorganism integrated fixed CO 2 System, method and use for synthesizing single cell proteins |
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