CN117363551A - Method for increasing concentration of ethanol produced by Clostridium ljungdahlii synthesis gas fermentation and microbial cells - Google Patents
Method for increasing concentration of ethanol produced by Clostridium ljungdahlii synthesis gas fermentation and microbial cells Download PDFInfo
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 title claims abstract description 168
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- 238000000855 fermentation Methods 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 32
- 241000186566 Clostridium ljungdahlii Species 0.000 title claims abstract description 29
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- 239000013612 plasmid Substances 0.000 claims abstract description 59
- 101150059172 AOR gene Proteins 0.000 claims abstract description 25
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- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 2
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- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
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Abstract
A method of increasing the concentration of Clostridium ljungdahlii syngas fermentatively produced ethanol and microbial cells comprising the steps of: (1) Designing gRNA near an AOR gene promoter and synthesizing the sequence fragment; (2) constructing a CRISPRa activating fusion protein; (3) constructing a CRISPRa plasmid; (4) bacterial transformation and plasmid extraction; (5) target microbial transformation; (6) recovering and screening the strain; (7) And (3) fermenting and producing ethanol and microbial cells by using the strain obtained in the step (6) by adopting synthesis gas. The present application, by activating the AOR gene by CRISPRa, can achieve higher ethanol yields in syngas fermentation and further promote an increase in cell concentration by overproduction of ATP.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a method for improving the concentration of ethanol produced by Clostridium ljungdahlii synthesis gas fermentation and microbial cells.
Background
Anaerobic bacteria Clostridium ljungdahlii is a potential microbial plant and is widely studied for use in the fields of synthetic biology and metabolic engineering. It is capable of utilizing synthesis gas (containing CO and H in the absence of oxygen 2 Is used as a raw material for fermentation to produce valuable compounds such as ethanol, acetic acid and other biochemical products; in addition, the bacteria can be used for converting organic wastes into valuable compounds, thereby realizing the utilization of waste resources and helping to reduce carbon emission.
Anaerobe Clostridium ljungdahlii characteristics: such bacteria grow in an anoxic environment and are therefore useful in particular manufacturing processes such as waste treatment and biofuel production; the method has unique gas fermentation capability, and can utilize synthesis gas as a carbon source and an energy source to synthesize the organic compound through multi-step reaction. Clostridium ljungdahlii has great potential as an anaerobic bacterium in the fermentation of synthesis gas to produce compounds such as ethanol, acetic acid, and the like.
Overall, when carbon monoxide is used as a feedstock to produce ethanol by syngas fermentation, the chemical reaction formula is as follows: 6CO+3H 2 O→C 2 H 5 OH+4CO 2 I.e., six molecules of carbon monoxide react with three molecules of water to produce ethanol. When using carbon monoxide as a feedstock, acetic acid is produced by syngas fermentation, the chemical reaction formula is: 4CO +2H 2 O→CH 3 COOH+2CO 2 I.e., four molecules of carbon monoxide react with two molecules of water to form acetic acid. In these reactions, carbon monoxide (CO) in the synthesis gas reacts catalytically with anaerobic bacteria Clostridium ljungdahlii under specific metabolic pathways to produce useful compounds such as acetic acid and ethanol. It should be noted, however, that these pathways may vary among anaerobic bacteria Clostridium ljungdahlii depending on different conditions and circumstances, and may also involve the action of other intermediate metabolites and accessory enzymes.
The metabolic Pathway used for anaerobic fermentation of synthesis gas is the Acetyl-CoA metabolic Pathway (Acetyl-CoA Pathway), also known as the Wood-Ljungdahl Pathway (WL Pathway), and details are shown in FIG. 1. In this pathway, anaerobic fermentation of synthesis gas typically utilizes acetyl-CoA as an intermediate metabolite to synthesize compounds such as acetic acid and ethanol via different metabolic pathways. This is a complex metabolic network involving multiple enzymatic steps.
Among the possible metabolic pathways for the synthesis of acetic acid are: carbon monoxide and formic acid (Formate) are converted to acetyl-CoA, and then acetic acid is synthesized by a subsequent reaction. The carbon monoxide is hydrogenated to formic acid, which is then oxidized to CO 2 And CO 2 Synthesizing acetic acid with hydrogen. This is a key metabolic pathway involving the synergistic action of multiple enzymes.
For ethanol synthesis, there are two possible metabolic pathways: (1) Similar to acetate synthesis, acetyl-CoA produced in the WL pathway is first converted to acetate, which process generates a molecule of ATP, and then the intermediate acetate is converted to acetaldehyde and then to ethanol by a subsequent enzyme catalysis step. (2) In some cases, ethanol synthesis may also be accomplished by reducing acetyl-CoA to directly form acetaldehyde, which then forms ethanol.
It should be noted that in the two different ethanol production processes, there are different ATP balances and energy balances. In pathway (1), the system produces an additional ATP with the production of each acetic acid molecule; in pathway (2), however, the production of ethanol does not produce additional ATP. The cell growth concentration (cell biological) of the strain Clostridium ljungdahlii was related to the ATP concentration. Thus, it is expected that the distribution of acetic acid and ethanol in different proportions in the process will affect the ATP output, which will necessarily affect the amount of cellular biomass. In summary, using acetyl-CoA as an intermediate metabolite, anaerobic fermentation of synthesis gas can produce valuable compounds such as acetic acid and ethanol, and also can accumulate microbial proteins, i.e., the microorganisms themselves.
The product ratio of the anaerobic fermentation of synthesis gas depends on a number of factors including the choice of strain, culture conditions, nutrient supply, gas flow and mass transfer etc. The metabolic pathways and growth conditions of the microorganism have an important influence on the distribution of the product. Under different environmental and operational strategies, microorganisms may modulate their metabolic pathways, resulting in a change in the product ratio. For example, if the microorganism is more prone to convert acetyl-CoA to ethanol, then ethanol production will dominate. Conversely, if the microorganism uses more acetyl-CoA for acetate synthesis, acetate may become the major product and the concentration of microbial cells may increase accordingly because of more ATP production. If more acetic acid is produced, the ethanol yield of the whole process is increased and the cell concentration is also increased through the metabolic pathway of acetic acid-acetaldehyde-ethanol to convert the acetic acid into ethanol.
The present application seeks to find a way to make the products as high value ethanol and microbial proteins as possible, rather than acetic acid, in terms of the production of the products acetic acid, ethanol, and microbial proteins of the synthesis gas fermentation. If the ethanol and protein are compared further, it is desirable that more of the product is microbial protein.
Disclosure of Invention
The purpose of the application is to provide a method for improving the concentration of Clostridium ljungdahlii synthetic gas fermentation ethanol production and microbial cells.
The technical scheme adopted for solving the technical problems is as follows:
a method of increasing the concentration of Clostridium ljungdahlii syngas fermentatively produced ethanol and microbial cells comprising the steps of:
(1) Designing gRNA near an AOR gene promoter and synthesizing the sequence fragment;
(2) Constructing a CRISPRa activated fusion protein;
(3) Constructing a CRISPRa plasmid;
(4) Bacterial transformation and plasmid extraction;
(5) Target microbial transformation;
(6) Recovering and screening the strain;
(7) And (3) fermenting and producing ethanol and microbial cells by using the strain obtained in the step (6) by adopting synthesis gas.
Further, the sequence segments in the step (1) are as follows:
forward gRNA sequence: 5'-CGCGTCTGCGAGCGCGGCAG-3'
Reverse gRNA sequence: 5'-CTGCCGCGCTCGCACGCGAC-3'.
Further, the construction of the CRISPRa activating fusion protein described in step (2): the use of dCAS9-VP64 as a basis for activating the fusion protein, when combined with gRNA, leads precisely to the targeting of the fusion protein to the promoter region of the target gene.
Further, the method of the CRISPRa plasmid member in step (3) is as follows: the CRISPRa plasmid was constructed by inserting the gRNA sequence synthesized in step (1) and the activation fusion protein gene constructed in step (2) into a plasmid using pUC19 as a basic plasmid backbone.
Further, the method of bacterial transformation and plasmid extraction described in step (4): introducing the constructed CRISPRa plasmid into a proper E.coli (E.coli) cell, culturing the E.coli, and carrying out plasmid amplification; the synthetic CRISPRa plasmid was then extracted from e.coli cells and prepared for transformation of the target microorganism.
Further, the target microorganism in step (5) is transformed: the constructed CRISPRa plasmid is introduced into target microorganism Clostridium ljungdahlii (CL-wild) and is transformed and constructed by an electrotransformation method.
Further, the specific steps of the target microbial transformation include:
bacterial culture: culturing CL-wild to logarithmic phase before electrotransformation, wherein the concentration is OD600 of about 0.5;
pretreatment of cells: centrifuging and washing the cultured bacteria to obtain a proper amount of bacteria, so that the bacteria are in an optimal state;
plasmid preparation: extracting the constructed CRISPRa plasmid;
electric conversion: mixing the prepared plasmid with bacterial cells, then placing the mixture in an electrotransformation instrument, and introducing the plasmid into the cells through electric field pulse;
and (5) recovering: after electrotransformation, the bacteria are restored in an appropriate medium, gradually restored and expression of the target gene is started during the restoration period;
screening: screening is performed on appropriate media to identify bacterial strains harboring the CRISPRa plasmid, based on the selection genes on the plasmid.
Further, the electric conversion conditions are as follows:
electric field strength: the electric field intensity suitable for Clostridium ljungdahlii is adopted within the range of 20 kV/cm;
electroporation pulse number: a pulse number of 2-15 is used to ensure that enough cells are electrically transformed;
the components of the conversion solution are as follows: tris-HCl buffer, including 10% PEG, and 5mM calcium chloride, was used at a concentration of 50 mM; 50ng/uL of CRISPRa plasmid was added to the transformation solution;
glucose was added at 0.5% and peptone at 0.5% was used as a carbon source and a nitrogen source required for cell growth.
Recovery conditions after conversion: after electrotransformation, the bacteria need to be recovered in an appropriate medium. The culture conditions during recovery may be optimized according to the characteristics of the microorganism.
Further, recovering and screening the strain described in step (6): after electrotransformation, the bacteria need to be recovered in an appropriate medium; and (3) selecting the reinforced clostridium culture medium to culture microorganisms under strict anaerobic conditions to obtain the strain CL-P1006 which improves the ethanol yield and the cell concentration by utilizing the fermentation of the synthesis gas.
Careful investigation of the metabolic pathways leading to acetic acid and to acetaldehyde and ethanol, it was found that there is one of the most critical enzymes AOR, which catalyzes the conversion of acetic acid to acetaldehyde and thus ethanol. In the metabolic step of acetic acid to acetaldehyde, AOR represents an Acetyl-CoA Decarbonylase/Acetyl-CoA Synthase complex enzyme. This complex enzyme plays a key role in the anaerobic fermentation of synthesis gas, and its main function is to catalyze the conversion of acetic acid, producing acetaldehyde and CoA. This process involves two key steps, decarboxylation and CoA synthesis, acetaldehyde being an intermediate in the subsequent synthesis of products such as ethanol. AOR complex enzymes are typically composed of multiple genes encoding different subunits in the genome of a microorganism. Wherein the alpha subunit (CtfA) acts as a decarboxylase in the complex enzyme, removing the carboxyl group (carbonyl group) from the acetic acid to form an intermediate product, which can be further converted to acetaldehyde. The additional β subunit (CtfB) acts as a CoA synthase in a complex enzyme, which binds one CoA to the carbon backbone in the decarboxylated acetate to form acetaldehyde and CoA. Using the method of CRISPR-Cas9 gene editing, an activating fusion protein can be constructed by designing specific grnas, and inserting the synthesized gRNA sequence and activating fusion protein gene into a plasmid to construct a CRISPRa plasmid. The constructed plasmid is guided into E.coli (E.coli) cells, extracted after plasmid amplification, then guided into target microorganism Clostridium ljungdahlii by means of electrotransformation, and then strain with high expression of AOR is obtained by screening, and then synthesis gas fermentation verification is carried out by a fermentation tank.
Compared with the prior art, the invention has the following beneficial effects:
the application can significantly improve ethanol yield during syngas fermentation by activating the AOR gene using the CRISPRa technique. Activation of the AOR gene results in increased expression of acetaldehyde dehydrogenase (AOR enzyme), promoting the reductive conversion of acetaldehyde to ethanol, thereby increasing the rate of ethanol production. Furthermore, since AOR enzyme requires NADH as a co-reducing agent, activation of AOR gene may lead to an increase in ATP production, thereby providing more reducing power and further promoting ethanol synthesis.
The CRISPRa activation of the AOR gene not only can increase the ethanol yield, but also can cause the increase of the intracellular ATP and NADH concentration, thereby affecting the cell concentration. Thus, by activating the AOR gene, the dual advantages of ethanol production by syngas fermentation, i.e., increased ethanol production and increased cell concentration, can be achieved.
Thus, by activating the AOR gene by CRISPRa, higher ethanol yields can be achieved in syngas fermentation and further increase in cell concentration is promoted by overproduction of ATP. The strategy is expected to bring new breakthrough for ethanol production in the field of microbial fermentation engineering and provide powerful support for sustainable energy production.
Drawings
FIG. 1 is a schematic diagram of the acetyl-CoA metabolic pathway.
Detailed Description
The technical scheme of the invention is further described below with reference to specific embodiments.
A method for increasing the concentration of ethanol and microbial cells produced by the fermentation of Clostridium ljungdahlii syngas, comprising the steps of:
(1) Designing gRNA near an AOR gene promoter and synthesizing the sequence fragment;
(2) Constructing a CRISPRa activated fusion protein;
(3) Constructing a CRISPRa plasmid;
(4) Bacterial transformation and plasmid extraction;
(5) Target microbial transformation;
(6) Recovering and screening the strain;
(7) And (3) fermenting and producing ethanol and microbial cells by using the strain obtained in the step (6) by adopting synthesis gas.
Further, the gRNA in the vicinity of the AOR gene promoter was designed and the sequence fragment was synthesized as described in step (1), and the following sequence was highly specific and active, although there were various choices. Target area: vicinity of AOR Gene promoter
Forward gRNA sequence: 5'-CGCGTCTGCGAGCGCGGCAG-3'
Reverse gRNA sequence: 5'-CTGCCGCGCTCGCACGCGAC-3'.
In this step, localization of the AOR gene: first, it is necessary to determine the location and role of the AOR gene in the Clostridium ljungdahlii genome. This ensures accurate targeting to the upstream region of the AOR gene when designing gRNA to achieve an effective CRISPRa effect. Design of gRNA: the design of a suitable gRNA is critical for successful CRISPRa realization. The gRNA should be precisely targeted to the upstream promoter region of the AOR gene to ensure activation.
Further, the construction of the CRISPRa activating fusion protein described in step (2): to achieve CRISPRa, the appropriate activating fusion protein needs to be selected. The activation fusion protein should be selected to be effective in enhancing the transcriptional activity of the target gene in the target microorganism. In this example, dCAS9-VP64 was used as the basis for activating the fusion protein, which, when combined with gRNA, could precisely direct the activation of the fusion protein to the promoter region of the target gene.
Further, the CRISPRa plasmid was constructed as described in step (3), the expression of the activating fusion protein was combined with the gRNA and integrated into the appropriate plasmid backbone. The CRISPRa plasmid building block method is: the CRISPRa plasmid was constructed by inserting the gRNA sequence synthesized in step (1) and the activation fusion protein gene constructed in step (2) into a plasmid using pUC19 as a basic plasmid backbone.
Further, the method of bacterial transformation and plasmid extraction described in step (4): introducing the constructed CRISPRa plasmid into a proper escherichia coli (E.coli) cell, culturing the escherichia coli for 48 hours at 30 ℃ and 100rpm, and carrying out plasmid amplification; the synthetic CRISPRa plasmid was then extracted from e.coli cells and prepared for transformation of the target microorganism.
Further, the target microorganism in step (5) is transformed: the constructed CRISPRa plasmid is introduced into target microorganism Clostridium ljungdahlii (CL-wild) and is transformed and constructed by an electrotransformation method. The method comprises the following specific steps:
bacterial culture: culturing CL-wild to logarithmic phase before electrotransformation, wherein the concentration is OD600 of about 0.5;
pretreatment of cells: centrifuging and washing the cultured bacteria to obtain a proper amount of bacteria, so that the bacteria are in an optimal state;
plasmid preparation: extracting the constructed CRISPRa plasmid;
electric conversion: mixing the prepared plasmid with bacterial cells, then placing the mixture in an electrotransformation instrument, and introducing the plasmid into the cells through electric field pulse;
and (5) recovering: after electrotransformation, the bacteria are restored in an appropriate medium, gradually restored and expression of the target gene is started during the restoration period;
screening: screening is performed on appropriate media to identify bacterial strains harboring the CRISPRa plasmid, based on the selection genes on the plasmid.
In this example, the following electrotransformation conditions were employed:
electric field strength: the electric field intensity suitable for Clostridium ljungdahlii is adopted within the range of 20 kV/cm;
electroporation pulse number: a pulse number of 2-15 is used to ensure that enough cells are electrically transformed;
the components of the conversion solution are as follows: tris-HCl buffer, including 10% PEG, and 5mM calcium chloride, was used at a concentration of 50 mM; 50ng/uL of CRISPRa plasmid was added to the transformation solution;
glucose was added at 0.5% and peptone at 0.5% was used as a carbon source and a nitrogen source required for cell growth.
Recovery conditions after conversion: after electrotransformation, the bacteria need to be recovered in an appropriate medium. The culture conditions during recovery may be optimized according to the characteristics of the microorganism.
Further, recovering and screening the strain described in step (6): after electrotransformation, the bacteria need to be recovered in an appropriate medium; and (3) selecting the reinforced clostridium culture medium to culture microorganisms under strict anaerobic conditions to obtain the strain CL-P1006 which improves the ethanol yield and the cell concentration by utilizing the fermentation of the synthesis gas.
Further, ethanol production studies using the strains CL-wild and CL-P1006 syngas:
two different groups of strains CL-wild and transformed strain CL-P1006 were cultivated using an enhanced Clostridium medium, and incubated in a sealed plasma bottle under strictly anaerobic conditions for 72 hours, the bottle being filled with CO gas at a pressure of 0.5 kg, the shaking table setting temperature being 37℃and 200rpm. After every 12 hours, the old gas was withdrawn and fresh CO gas was re-injected into the 0.5 kg column.
Measuring Acetic Acid (AA), ethanol (EtOH), and strain concentration (OD) at the time of inoculation, i.e., at 0 hours; acetic acid, ethanol, and strain concentrations were measured at the end of the 72 hour experiment. The results are shown in the following table:
clearly, the CL-P1006 strain promotes more ethanol production, and more cell production, by over-expressing AOR than the strain CL-wild without CRISPRa activation of the AOR gene.
The above embodiments are merely illustrative of the preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, but various modifications and improvements made by those skilled in the art to which the present invention pertains will be made without departing from the spirit of the present invention, and it is intended to fall within the scope of the present invention as defined in the appended claims.
Claims (9)
1. A method for increasing the concentration of ethanol and microbial cells produced by the fermentation of Clostridium ljungdahlii syngas, comprising the steps of:
(1) Designing gRNA near an AOR gene promoter and synthesizing the sequence fragment;
(2) Constructing a CRISPRa activated fusion protein;
(3) Constructing a CRISPRa plasmid;
(4) Bacterial transformation and plasmid extraction;
(5) Target microbial transformation;
(6) Recovering and screening the strain;
(7) And (3) fermenting and producing ethanol and microbial cells by using the strain obtained in the step (6) by adopting synthesis gas.
2. The method of increasing the concentration of ethanol and microbial cells produced by fermentation of Clostridium ljungdahlii syngas according to claim 1, wherein the sequence segments in step (1) are:
forward gRNA sequence: 5'-CGCGTCTGCGAGCGCGGCAG-3'
Reverse gRNA sequence: 5'-CTGCCGCGCTCGCACGCGAC-3'.
3. The method of increasing the concentration of ethanol and microbial cells produced by fermentation of Clostridium ljungdahlii syngas according to claim 1, wherein the constructing a CRISPRa activating fusion protein in step (2): the use of dCAS9-VP64 as a basis for activating the fusion protein, when combined with gRNA, leads precisely to the targeting of the fusion protein to the promoter region of the target gene.
4. The method of claim 1, wherein the CRISPRa plasmid means in step (3) is constructed by: the CRISPRa plasmid was constructed by inserting the gRNA sequence synthesized in step (1) and the activation fusion protein gene constructed in step (2) into a plasmid using pUC19 as a basic plasmid backbone.
5. The method for increasing the concentration of ethanol and microbial cells produced by fermentation of Clostridium ljungdahlii syngas according to claim 1, wherein the method for bacterial transformation and plasmid extraction in step (4) comprises: introducing the constructed CRISPRa plasmid into an escherichia coli cell, culturing the escherichia coli, and carrying out plasmid amplification; the synthetic CRISPRa plasmid was then extracted from e.coli cells and prepared for transformation of the target microorganism.
6. The method of increasing the concentration of Clostridium ljungdahlii syngas fermentative production of ethanol and microbial cells of claim 1, wherein said target microbial conversion in step (5): the constructed CRISPRa plasmid is introduced into target microorganism Clostridium ljungdahlii, and is transformed and constructed by an electrotransformation method.
7. The method of increasing the concentration of ethanol and microbial cells produced by fermentation of Clostridium ljungdahlii syngas in accordance with claim 6, wherein said specific step of target microbial conversion comprises:
bacterial culture: culturing CL-wild to logarithmic phase before electrotransformation;
pretreatment of cells: centrifuging and washing the cultured bacteria to obtain a proper amount of bacteria, so that the bacteria are in an optimal state;
plasmid preparation: extracting the constructed CRISPRa plasmid;
electric conversion: mixing the prepared plasmid with bacterial cells, then placing the mixture in an electrotransformation instrument, and introducing the plasmid into the cells through electric field pulse;
and (5) recovering: after electrotransformation, the bacteria are restored in the medium, gradually restored during the restoration period and the expression of the target gene is started;
screening: screening is performed on the medium to identify bacterial strains harboring the CRISPRa plasmid, based on the selection gene on the plasmid.
8. The method of increasing the concentration of Clostridium ljungdahlii syngas fermentation produced ethanol and microbial cells of claim 7, wherein said electrotransformation conditions:
electric field strength: the electric field intensity suitable for Clostridium ljungdahlii is adopted within the range of 20 kV/cm;
electroporation pulse number: adopting the pulse number of 2-15;
the components of the conversion solution are as follows: tris-HCl buffer, including 10% PEG, and 5mM calcium chloride, was used at a concentration of 50 mM; 50ng/uL of CRISPRa plasmid was added to the transformation solution;
glucose was added at 0.5% and peptone at 0.5% was used as a carbon source and a nitrogen source required for cell growth.
9. The method of increasing the concentration of Clostridium ljungdahlii syngas fermentative production of ethanol and microbial cells of claim 1, wherein the recovering and selecting strain of step (6): after electrotransformation, bacteria need to be recovered in the medium; and (3) selecting the reinforced clostridium culture medium to culture microorganisms under strict anaerobic conditions to obtain the strain CL-P1006 which improves the ethanol yield and the cell concentration by utilizing the fermentation of the synthesis gas.
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