Patent Grant
11214816
This application is a U.S. national phase under the provisions of 35 U.S.C. § 371 of International Patent Application No. PCT/KR17/13171 filed Nov. 20, 2017, which in turn claims priority of Korean Patent Application No. 10-2016-0180223 filed Dec. 27, 2016 and Korean Patent Application No. 10-2017-0151642 filed Nov. 14, 2017. The disclosures of such international patent application and Korean priority patent applications are hereby incorporated herein by reference in their respective entireties, for all purposes.
The present invention relates to a recombinant microorganism introduced with heterologous genes and a method for producing a useful substance from formic acid and carbon dioxide using the microorganism. More specifically, the present invention relates to a recombinant microorganism, which is capable of efficiently synthesizing one molecule of pyruvate by introducing heterologous genes into the microorganism having a cyclic metabolic pathway and simultaneously assimilating two molecules of formic acid and one molecule of carbon dioxide, and a method for producing a useful substance from formic acid and carbon dioxide using the microorganism.
In order to develop carbon fixation technologies to mitigate climate change, researches are actively underway to convert a compound consisting of one carbon, such as carbon dioxide into an organic substance having a higher carbon number. Researches on the conversion of compounds consisting of one carbon (a C1 compound) can be roughly classified into chemical methods and biological methods. Among them, chemical methods are carried out using electrochemical reactions based on metal- and non-metal-catalysts. Through such reactions, carbon dioxide is converted into a non-gaseous carbon compound such as methanol and formic acid, and examples of related research include conversion using a carbon nanotube catalyst (Kumar et al., Nat. Comm. 2819, 2013), conversion using an iron catalyst (Christopher et al., Angew. Chem. Int. Ed. 49:50, 9777-9780, 2010) and conversion using an alloy catalyst (Studt et al., Nat. Chem., 6, 320-324, 2014) and the like. Conversion using these chemical methods has an advantage of a relatively highspeed, but has a limitation in that the products obtained through the C1 compound conversion process are single-carbon substances such as formic acid and methanol, rather than useful compounds containing multiple carbon atoms.
Regarding biological conversion methods, researches are underway with the goal of converting a gaseous C1 compound into a non-gaseous compound and converting a C1 compound into a useful compound consisting of several carbon atoms. In the former case, researches have been conducted on the use of metabolic pathways present in nature and the improvement thereof (PCT/US2008/083056), designs of new metabolic pathways (Schwander et al., Science, 354: 6314, 900-904, 2016) and the like. Examples of the latter case include the production of useful compounds using methanol-assimilating microorganisms (US 2003/0124687 A1), the production of compounds having three carbon atoms from formic acid (US 2013/0196359 A1) and the like. However, these researches have limitations in that it is difficult to identify the effects thereof under internal conditions of organisms due to the low efficiency thereof and in that whether they can function in a manner compatible with native metabolic pathways in living organisms has not been verified.
Therefore, research to overcome the limitations of the biological C1 compound conversion process described above and to thereby develop an efficient C1 compound conversion process is arising in the industry. In particular, among the C1 compounds, formic acid has benefits in that it is relatively less toxic to organisms than other C1 compounds and is advantageous for assimilation (anabolic) reaction compared to C1 compounds in terms of reaction kinetics, and can be synthesized easily and rapidly from carbon dioxide through chemical methods. However, the genetic information of the genus Methylobacterium, which is a representative formic acid-assimilating bacterium, was reported only in 2009, that is to say, basic research for application thereof was only conducted relatively recently, and research on the conversion of formic acid is still at an early stage. At present, research relating to assimilation (anabolism) of formic acid is still at the level of the presentation and designing of metabolic pathways using an enzyme information database (US 2015/0218528 A1; Arren Bar-Even., Biochemistry, 55:28, 3851-3863), and no case demonstrating the same has been reported yet.
Accordingly, the present inventors have designed a novel metabolic pathway for converting a C1 compound, formic acid, into a useful compound having a plurality of carbon atoms, and have attempted to demonstrate the metabolic pathway under in vivo conditions of organisms. As a result, the present inventors have developed a cyclic metabolic pathway which involves effectively assimilating formic acid and carbon dioxide at the same time, thereby producing a single molecule of acetyl-CoA as a product of the cyclic metabolic pathway, when introducing novel genes derived from the genus Methylobacterium, the genus Rhodobacter, the genus Chloroflexus, the genus Acetobacterium, the genus Roseobacter and the genus Mannheimia, and have identified that acetyl-CoA obtained as the product of the cyclic metabolic pathway binds to a single molecule of formic acid to produce pyruvate, as a C3 compound, when further introducing a reverse reaction of pyruvate formate lyase. Further, the present inventors have developed a metabolic pathway that is capable of producing glycine, serine and pyruvate by effectively assimilating formic acid and carbon dioxide using novel genes derived from the genus Methylobacterium microorganism and genes derived from the genus Escherichia microorganism and have identified that glycine, serine and pyruvate were produced from formic acid and carbon dioxide using the corresponding metabolic pathway. Based on these findings, the present invention has been completed.
Therefore, it is one object of the present invention to provide a recombinant microorganism for producing a useful substance including a C3 compound from formic acid and carbon dioxide.
It is another object of the present invention to provide a method for producing a useful substance including a C3 compound from formic acid and carbon dioxide using the recombinant microorganism.
In accordance with one aspect of the present invention, provided is a recombinant microorganism obtained by introducing a gene encoding an enzyme involved in a formic acid assimilation pathway or a recombinant vector containing the gene into a host microorganism having a central carbon assimilation pathway.
In accordance with another aspect of the present invention, provided is a method for producing a useful compound having a C3 compound as an intermediate product including: (a) culturing the recombinant microorganism with formic acid and carbon dioxide as a carbon source to produce a useful substance having a C3 compound as an intermediate product; and (b) recovering the resulting useful substance.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Reference will now be made in detail to the preferred embodiments of the present invention, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as appreciated by those skilled in the field to which the present invention pertains. In general, the nomenclature used herein is well-known in the art and is ordinarily used.
The present inventors first experimentally identified a novel cyclic metabolic pathway capable of synthesizing compounds composed of three or more carbon atoms from formic acid and carbon dioxide. That is, the present inventors designed the cyclic metabolic pathways shown in
Specifically, the present inventors constructed a novel cyclic metabolic pathway for assimilating formic acid and have identified that formic acid was assimilated through the metabolic pathway when the metabolic pathway was introduced into the host microorganism. In addition, the present inventors have found that the assimilation efficiency of formic acid was significantly increased when using some enzymes involved in the metabolic pathway introduced from exotic microorganisms (
Therefore, in one aspect, the present invention is directed to a recombinant microorganism obtained by introducing a gene encoding an enzyme involved in a formic acid assimilation pathway or a recombinant vector containing the gene into a host microorganism having a central carbon assimilation pathway.
In the present invention, the central carbon assimilation pathway is a cyclic pathway for fixing carbon dioxide in microorganisms. Genes, coenzymes and energy transfer substances involved in the central carbon assimilation pathway of E. coli are shown in
In the present invention, the formic acid assimilation pathway can be combined with the central carbon assimilation pathway to synthesize a C3 or larger carbon compound. The host microorganism inherently possesses a central carbon assimilation pathway i); or has a central carbon assimilation pathway introduced externally ii).
The enzyme involved in the formic acid assimilation pathway in the present invention may be a gene of any one selected from the group consisting of formate-tetrahydrofolate ligase, methenyl tetrahydrofolate cyclohydrolase and methylene-tetrahydrofolate dehydrogenase, but is not limited thereto.
In the present invention, the genes may be derived from any one selected from the group consisting of Methylobacterium, Roseobacter, Rhodobacter, Chloroflexus, Acetobacterium, Mannheimia, Escherichia and Arabidopsis, but are not limited thereto.
In the present invention, the gene encoding formate-tetrahydrofolate ligase may be a nucleic acid molecule represented by SEQ ID NO: 7, the gene encoding methenyl tetrahydrofolate cyclohydrolase may be a nucleic acid molecule represented by SEQ ID NO: 8, and the gene encoding methylene-tetrahydrofolate dehydrogenase may be a nucleic acid represented by SEQ ID NO: 9 or SEQ ID NO: 19, but is not limited thereto.
Meanwhile, the present inventors have verified whether or not formic acid assimilated by the formic acid assimilation pathway was assimilated into glycolysis, which is one of the central carbon metabolic processes of the host microorganism. Also the present inventors have found that assimilation efficiency was improved when substituting a part of genes involved in the main carbon metabolism with heterologous genes (
Accordingly, in another aspect, the present invention is directed to a recombinant microorganism that has an improvement in a central carbon assimilation pathway of a host microorganism.
In the present invention, at least one enzyme involved in the central carbon assimilation pathway and selected from the group consisting of serine-glyoxylate aminotransferase, malyl-CoA lyase, malate-CoA ligase, succinyl-CoA:(S)-malate CoA-transferase, phosphoenolpyruvate carboxykinase, and hydroxypyruvate reductase may be substituted, further introduced or amplified but is not limited thereto.
The substituted or further introduced enzymes may be enzymes derived from any one selected from the group consisting of Methylobacterium, Roseobacter, Rhodobacter, Chloroflexus, Acetobacterium, Mannheimia, Escherichia and Arabidopsis, but are not limited thereto.
In the present invention, the enzyme involved in the central carbon assimilation pathway may be substituted with one of the following:
i) a serine-glyoxylate transaminase encoded by a nucleic acid molecule selected from the group consisting of a nucleic acid molecule represented by SEQ ID NO: 6, a nucleic acid molecule represented by SEQ ID NO: 13, a nucleic acid molecule represented by SEQ ID NO: 16, and a nucleic acid molecule represented by SEQ ID NO: 21;
ii) malyl-CoA lyase encoded by a nucleic acid molecule selected from the group consisting of a nucleic acid molecule represented by SEQ ID NO: 5, a nucleic acid molecule represented by SEQ ID NO: 14, and a nucleic acid molecule represented by SEQ ID NO: 15;
iii) malate-CoA ligase encoded by a nucleic acid molecule selected from the group consisting of a nucleic acid molecule represented by SEQ ID NO: 3, a nucleic acid molecule represented by SEQ ID NO: 4, a nucleic acid molecule represented by SEQ ID NO: 11, and a nucleic acid molecule represented by SEQ ID NO: 12;
iv) succinyl-CoA:(S)-malate CoA-transferase encoded by a nucleic acid molecule represented by SEQ ID NO: 17 or a nucleic acid molecule represented by SEQ ID NO: 18;
v) phosphoenolpyruvate carboxykinase encoded by a nucleic acid molecule represented by SEQ ID NO: 20; and/or
vi) hydroxypyruvate reductase encoded by a nucleic acid molecule of SEQ ID NO: 10, but is not limited thereto.
In one embodiment of the present invention, regarding the enzyme, the enzyme involved in the central carbon assimilation pathway of the Escherichia genus shown in
In the present invention, the genes may be transformed into a host microorganism in the form of a plasmid containing a synthetic operon (see
Meanwhile, when the formic acid assimilation pathway is combined with the central carbon assimilation pathway of the host microorganism, carbon dioxide and formic acid are converted into acetyl-CoA. The present inventors have found that formic acid could be further assimilated to produce a C3 compound by further introducing a metabolic pathway based on pyruvate formate lyase (
Thus, in another aspect, the present invention is directed to a recombinant microorganism having a C3 compound synthesis metabolic pathway, in which the formic acid assimilation pathway is combined with a central carbon assimilation pathway and a pyruvate formate lyase is further introduced thereto.
In the present invention, the recombinant microorganism may be further introduced with a gene encoding pyruvate formate lyase or a recombinant vector containing the gene.
In the present invention, the gene encoding pyruvate formate lyase may be a nucleic acid molecule represented by SEQ ID NO: 22, but is not limited thereto.
Another type of central carbon assimilation pathway developed in the present invention is a pathway for fixing formic acid and carbon dioxide in microorganisms, and
In the present invention, the formic acid and carbon dioxide assimilation pathways synthesize one molecule of glycine from carbon dioxide and formic acid, and synthesizes one molecule of serine from glycine and one molecule of formic acid. The synthesized serine is converted into pyruvate through serine deaminase and a C3 or longer compound can be synthesized from pyruvate. The host microorganism has the carbon assimilation capability by externally introducing the formic acid and carbon dioxide assimilation pathways.
That is, the present invention verified that serine and glycine can be synthesized from formic acid and carbon dioxide via the formic acid and carbon dioxide assimilation pathways, and it was confirmed that assimilation efficiency is improved through the change of the gene expression intensity and the deletion of a specific gene in the host microorganism (
In addition, the present invention verified that pyruvate can be synthesized from formic acid and carbon dioxide via the formic acid and carbon dioxide assimilation pathways, and it was confirmed that assimilation efficiency is improved through the change of the gene expression intensity and the deletion of a specific gene in the host microorganism (
In the present invention, the genes could be transformed into a host microorganism in the form of a plasmid containing a synthetic operon (
The results of transformation of the plasmid containing the synthetic operon into the host microorganism showed that the recombinant microorganism into which such a novel metabolic pathway was introduced exhibited considerably improved capability to synthesize glycine, serine and pyruvate from formic acid and carbon dioxide.
Accordingly, in another aspect, the present invention is directed to a recombinant microorganism having a metabolic pathway for synthesizing a C3 compound wherein glycine, serine and pyruvate are synthesized from formic acid and carbon dioxide through the introduction of a novel metabolic pathway.
The present invention relates to a recombinant microorganism having improved assimilation from formic acid and carbon dioxide through introduction of a gene encoding an enzyme involved in a formic acid assimilation pathway or a recombinant vector containing the gene into a host microorganism having a central carbon assimilation pathway. The enzyme is at least one selected from the group consisting of formate-tetrahydrofolate ligase, methenyl tetrahydrofolate cyclohydrolase, and methylene-tetrahydrofolate dehydrogenase.
In the present invention, the gene encoding formate-tetrahydrofolate ligase is a nucleic acid molecule represented by SEQ. ID. NO: 7, the gene encoding methenyl tetrahydrofolate cyclohydrolase is a nucleic acid molecule represented by SEQ ID NO: 8, and the gene encoding methylene-tetrahydrofolate dehydrogenase is a nucleic acid molecule represented by SEQ ID NO: 9 or SEQ ID NO: 19, but the present invention is not limited thereto.
In the present invention, a glycine cleavage complex may be enhanced, amplified or further introduced into the recombinant microorganism, and expression of the gene constituting the glycine cleavage complex may be reinforced by substituting an intrinsic promoter with any one strong promoter selected from the group consisting of a trc promoter, a tac promoter, a T7 promoter, a lac promoter and a trp promoter, or is overexpressed through a plasmid overexpression system. The glycine cleavage complex may be a nucleic acid molecule represented by SEQ ID NO: 63, but is not limited thereto.
In one embodiment of the present invention, a recombinant microorganism that glycine cleavage complex is enhanced, amplified in or further introduced into is produced by overexpressing the glycine cleavage complex represented by SEQ ID NO: 63 through a plasmid overexpression system or substituting the intrinsic promoter located upstream of the gcvT gene of the glycine cleavage complex with a strong promoter {SEQ ID NO: 67 (NCBI information: NC_000913.3 Region 3049125-3050667 was substituted with SEQ ID NO: 68, and the changed sequence corresponds to the promoter}.
In the present invention, the recombinant microorganism may be characterized in that the gcvR gene is deleted, and the gcvR gene may be a nucleic acid molecule represented by SEQ ID NO: 66, but the present invention is not limited thereto.
In the present invention, serine diaminase may be enhanced, amplified or further introduced into the recombinant microorganism, and the serine deaminase may be a nucleic acid molecule represented by SEQ ID NO: 69, but the present invention is not limited thereto.
In the present invention, the host microorganism may inherently possess a central carbon assimilation pathway i); or may have a central carbon assimilation pathway introduced externally ii).
In the present invention, the genes may be derived from any one selected from the group consisting of the genera Methylobacterium, Roseobacter, Rhodobacter, Chloroflexus, Acetobacterium, Mannheimia, Escherichia and Arabidopsis, but the present invention is not limited thereto.
In the present invention, the recombinant microorganism is capable of biosynthesizing pyruvate, glycine or serine assimilated from formic acid and carbon dioxide, but the present invention is not limited thereto.
In the present invention, the recombinant microorganism may be selected from the group consisting of the genera Escherichia, Mannheimia, Rhodobacter and Methylobacterium, but the present invention is not limited thereto.
The gene of the present invention may undergo variations in many ways in the coding region so long as the amino acid sequence of the protein expressed from the coding region is not changed, and may undergo variations or modifications so long as the expression of genes is not affected in a region excluding the coding region, and such varied or modified genes also fall within the scope of the present invention.
Therefore, the present invention also includes a polynucleotide having a base sequence substantially identical to the gene as well as a fragment of the gene. The term “substantially identical polynucleotide” means a gene encoding an enzyme having the same function as that used in the present invention, regardless of the homology of the sequence. The term “fragment of the gene” also means a gene encoding an enzyme having the same function as that used in the present invention, regardless of the length of the fragment.
In addition, the amino acid sequence of the protein, which is an expression product of the gene of the present invention, can be obtained from biological resources such as various microorganisms, so long as the titer and activity of the corresponding enzyme are not affected, and these biological resources also fall within the scope of the present invention.
Thus, the present invention also includes polypeptides having amino acid sequences substantially identical to the protein, as well as fragments of the polypeptides. The term “substantially identical polypeptide” means a protein having the same function as that used in the present invention regardless of the homology of the amino acid sequence. The term “fragment of the polypeptide” also means a protein having the same function as that used in the present invention, regardless of the length of the fragment.
As used herein, the term “vector” means a DNA product containing a DNA sequence operably linked to a regulation sequence capable of expressing DNA in a suitable host, and may be a plasmid, phage particle or a simple potential genome insert. Once the vector is transformed with an appropriate host, it may replicate and function independently of the genome of the host, or may often be integrated with the genome itself. Since the plasmid is the most commonly used type of vector, the terms “plasmid” and “vector” are sometimes used interchangeably throughout the specification of the present invention. For the purpose of the present invention, a plasmid vector is preferably used. A typical plasmid vector that can be used for this purpose includes (a) a replication origin to efficiently conduct replication so as to include several to several hundred plasmid vectors per host cell, (b) an antibiotic resistance gene to screen a host cell transformed with the plasmid vector and (C) a restriction enzyme cleavage site into which a foreign DNA fragment is inserted. Even if an appropriate restriction enzyme cleavage site is not present, the vector and foreign DNA can be easily ligated using a synthetic oligonucleotide adapter or a linker according to a conventional method. After ligation, the vector should be transformed into an appropriate host cell. Transformation can be easily carried out using a calcium chloride method or electroporation (Neumann, et al., EMBO J., 1: 841, 1982).
Expression vectors well-known in the art can be used as vectors for enhancing or overexpressing genes according to the present invention.
When a nucleotide sequence is aligned with another nucleotide sequence based on functional relation, it is “operably linked” thereto. This may be gene(s) and regulatory sequence(s) linked in such a way so as to enable gene expression when a suitable molecule (e.g., a transcriptional activator protein) is linked to the regulatory sequence(s). For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide, when expressed as a pre-protein involved in the secretion of the polypeptide; and a promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the sequence; or a ribosome-binding site is operably linked to a coding sequence when it affects the transcription of the sequence; or the ribosome-binding site is operably linked to a coding sequence when positioned to facilitate translation. Generally, “operably linked” means that the linked DNA sequence is in contact therewith, or a secretory leader is in contact therewith and is present in the reading frame. However, the enhancer need not be in contact. The linkage of these sequences is carried out by ligation (linkage) at convenient restriction enzyme sites. When no such site exists, a synthetic oligonucleotide adapter or a linker according to a conventional method is used. As is well known in the art, in order to increase the expression level of a transgene in a host cell, the gene should be operably linked to a transcriptional/translational expression regulation sequence that functions in a selected expression host. Preferably, the expression regulation sequence and the corresponding gene are included in one recombinant vector containing both a bacterial selection marker and a replication origin. When the host cell is a eukaryotic cell, the recombinant vector should further include a useful expression marker in the eukaryotic expression host.
The host cell transformed with the recombinant vector described above constitutes another aspect of the present invention. As used herein, the term “transformation” means introducing DNA into a host and allowing the DNA to be replicable by an extrachromosomal factor or chromosomal integration.
It should be understood that not all vectors function identically in expressing the DNA sequences of the present invention. Likewise, not all hosts function identically for the same expression system. However, those skilled in the art will be able to make appropriate selections from among a variety of vectors, expression regulation sequences and hosts without excessive burden of experimentation and without departing from the scope of the present invention. For example, selection of a vector should be carried out in consideration of a host because the vector should be replicated therein. The number of replications of the vector, the ability to control the number of replications, and the expression of other proteins encoded by the corresponding vector, such as the expression of antibiotic markers, should be also considered.
In addition, the gene introduced in the present invention may be introduced into the genome of a host cell and exist as a chromosomal factor. It will be apparent to those skilled in the art that even insertion of the gene into the genome of the host cell has the same effect as introducing the recombinant vector into the host cell.
Any host microorganism can be used as the host microorganism of the present invention without limitation and the host microorganism is preferably an Escherichia genus, Mannheimia genus, Rhodobacter genus or Methylobacterium genus microorganism.
The present inventors have also developed a metabolic pathway for synthesizing a C3 compound by combining the above-mentioned cyclic metabolic pathway with the reverse reaction of pyruvate formate lyase, and identified that the metabolic pathway enables formic acid and carbon dioxide to be effectively fixed into a C3 compound.
Therefore, in another aspect, the present invention is directed to a recombinant microorganism capable of producing a C3 compound, wherein a gene encoding pyruvate formate lyase or a recombinant vector containing the gene is further introduced into the recombinant microorganism.
In the present invention, the pyruvate formate lyase has a forward reaction activity of decomposing pyruvate into formate and acetyl-CoA as well as a reverse reaction activity thereof, and has excellent reverse reaction activity, with the conversion number of reverse reaction of 280 per sec, and previous research has demonstrated that pyruvate can be synthesized through the reverse reaction of the enzyme (Zelcbuch et al., Biochemistry, 55:17, 2423-2426, 2016). The pyruvate formate lyase is inherently found in E. coli as a host microorganism, but, when depending on inherent expression, it is difficult to maintain the expression amount above a predetermined level, since the expression of the enzyme is regulated by culturing conditions. Therefore, in order to synthesize a stable C3 compound, in the present invention, the enzyme is further introduced based on the plasmid, or is controlled to be overexpressed using a strong promoter.
In the present invention, the gene encoding pyruvate formate lyase may be a nucleic acid molecule represented by SEQ ID NO: 22.
In the present invention, the gene may be overexpressed by any one strong promoter selected from the group consisting of a trc promoter, a tac promoter, a T7 promoter, a lac promoter, a BBa23100 synthetic promoter and a trp promoter.
In another aspect, the present invention is directed to a method for producing a useful substance having a C3 compound as an intermediate product using the recombinant microorganism.
The method for producing the useful substance according to the present invention includes: (a) culturing the recombinant microorganism with formic acid and carbon dioxide as a carbon source to produce a useful substance having a C3 compound as an intermediate product; and (b) recovering the resulting useful substance. In the present invention, the C3 compound may be pyruvate. Examples of the useful substance include alcohols, amino acids, organic acids, alkenes and polymeric monomers. More specifically, the C3 compound includes, but is not limited to, straight or branched alcohols having 3 or more carbon atoms, isobutanol, propanol, hexanol, heptanol, octanol, nonanol, decanol, tert-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 2-methyl-2-butanol, putrescine, L-ornithine, arginine, polycyclic aromatic hydrocarbons (PAHs), polylactate, polylactate-co-glycolate, poly(2-hydroxyisovalerate-co-lactate), polyhydroxybutyrate (PHB), 4-hydroxybutyrate, biodiesel, gasoline, olefin, 5-aminovaleric acid, gamma-butyric acid, 3-hydroxypropionic acid, 3-aminopropionic acid, acrylic acid, 1,3-diaminopropane, caprolactam, threonine, valine, isoleucine, fumaric acid, malic acid, succinic acid, ceramide, astaxanthin, silybin, lycopene, lutein, retinol and the like. Pyruvate is an intermediate which is formed during the decomposition process of biomass including glycolipid and wood through glycolysis in most microorganism genera including the genus Escherichia. Pyruvate is a substance that is in center of carbon rearrangement reactions through native metabolic pathways in microorganisms and thus can be converted into all useful substances reported to be capable of being produced in microorganisms through the native metabolic pathways of microorganisms or additional metabolic pathways introduced from the outside, which will be apparent to those skilled in the art. Accordingly, the present invention includes all substances that can be synthesized using pyruvate produced by the recombinant microorganism according to the present invention.
In another aspect, the present invention is directed to a method for producing a C3 compound using the recombinant microorganism.
The method according to the present invention includes: (a) culturing the recombinant microorganism in formic acid and carbon dioxide as a carbon source to produce a C3 compound; and (b) recovering the produced C3 compound, wherein the C3 compound is pyruvate, serine or glycine, but is not limited thereto.
Hereinafter, the present invention will be described in more detail with reference to the following examples. However, it will be obvious to those skilled in the art that the following examples are provided only for illustration of the present invention and should not be construed as limiting the scope of the present invention.
In order to construct a formic acid and carbon dioxide assimilation pathway and to synthesize a C3 compound with high efficiency, necessary genes were introduced into Escherichia coli, which is a target microorganism, using a recombinant plasmid. The plasmid used for construction of the assimilation pathway was a p10099A plasmid including an Ampicillin resistance gene, a pBR322 replication origin, and the synthetic promoter BBa 23100. PCR was conducted using the plasmid as a template and the primers of SEQ ID NO: 1 and SEQ ID NO: 2, and the amplified gene fragment was then recovered and purified to prepare a gene fragment used as a plasmid backbone for the production of the recombinant plasmid.
The foreign gene fragments required for plasmid production were prepared by conducting PCR using the genomic DNA of microorganisms having the corresponding gene as a template and using a primer designed for amplification of the gene, and then collecting and purifying the amplified gene fragment, and the corresponding genes and primer sequences are shown in Tables 1 and 2 below.
Methylobacterium extorquens
Methylobacterium extorquens
Methylobacterium extorquens
Methylobacterium extorquens
Methylobacterium extorquens
Methylobacterium extorquens
Methylobacterium extorquens
Methylobacterium extorquens
Roseobacter denitrificans
Roseobacter denitrificans
Roseobacter denitrificans
Rhodobacter sphaeroides
Rhodobacter sphaeroides
Rhodobacter sphaeroides
Chloroflexus aurantiacus
Chloroflexus aurantiacus
Acetobacterium woodii
Mannheimia succiniciproducens
Arabidopsis thaliana
Escherichia. Coli.
Another plasmid used for constructing the assimilation pathway was a plasmid including a chloramphenicol resistance gene and a replication origin p15A and a synthetic promoter BBa_23100. PCR was conducted using the plasmid as a template and the primers of SEQ ID NO: 1 and SEQ ID NO: 2, and then the amplified gene fragment was recovered and purified to prepare a gene fragment used as a plasmid backbone for the production of the recombinant plasmid.
The foreign gene fragments required for plasmid production were prepared by conducting PCR using the genomic DNA of microorganisms having the corresponding gene as a template and using a primer designed for amplification of the gene, and then collecting and purifying the amplified gene fragment, and the corresponding genes and primer sequences are shown in Tables 3 and 4 below.
Escherichia coli.
Escherichia coli.
The plasmid backbone gene fragments and amplified gene fragments were amplified using the Gibson assembly method (Gibson et al., Nat. Methods, 6:5, 343-345, 2009), which is commonly used for the assembly of gene fragments, and each plasmid was constructed to contain one or more of the foreign genes set forth in Table 1 or Table 3 above. In addition, a recombinant strain wherein the gcvR gene (SEQ ID NO: 66, NCBI information: NC_000913.3, Region 2599906-2600478) is deleted and the promoter of the gene constituting the glycine cleavage complex is changed {SEQ ID NO: 67 (NCBI Information: NC_000913.3 Region 3049125-3050667) is substituted as shown in SEQ ID NO: 68}. The recombinant strain having a gene deletion and substitution was produced using a homologous recombination method commonly used in the art (Datsenko et al., PNAS, 97: 12, 6640-6645, 2000). Representative plasmids prepared in the present invention are shown in Table 5 and
The recombinant plasmid produced by the method described above was transformed into E. coli to prepare recombinant E. coli. The E. coli used in the present invention was E. coli DH5α (Invitrogen, USA), and transformation into E. coli was carried out using a chemical transformation method commonly used in the art. Representative recombinant E. coli produced in the present invention are shown in Table 6 below.
Carbon isotope analysis was performed to verify whether or not the recombinant E. coli produced in Example 1 could effectively assimilate formic acid and carbon dioxide. In order to verify the assimilation of formic acid, experiments were performed using wild-type Escherichia coli (DH5α WT) as a control group and recombinant Escherichia coli (DH5α THF) introduced with formate-tetrahydrofolate ligase encoded by a nucleic acid molecule represented by SEQ ID NO: 7, methenyl tetrahydrofolate cyclohydrolase encoded by a nucleic acid molecule represented by SEQ ID NO: 8, and methylene-tetrahydrofolate dehydrogenase encoded by a nucleic acid molecule represented by SEQ ID NO: 9 among the recombinant Escherichia coli produced in Example 1.
In order to verify whether or not the assimilated formic acid is linked to the central carbon assimilation pathway, experiments were performed using a recombinant Escherichia coli further introduced with serine-glyoxylate transaminase encoded by a nucleic acid molecule represented by SEQ ID NO: 6, 3, 16 or 21. Further, in order to verify whether or not the assimilated formic acid is linked to the central carbon assimilation pathway and effectively synthesizes acetyl-CoA together with carbon dioxide, experiments were performed using a recombinant microorganism introduced with malyl-CoA lyase encoded by a nucleic acid molecule of SEQ ID NO: 5 or 15, and further introduced with a malate-CoA ligase encoded by a nucleic acid molecule represented by SEQ ID NO: 3, 4, 11 or 12.
For the carbon isotope analysis, the control and experimental E. coli were cultured in M9 medium containing formate and bicarbonate ion labeled with a 13C carbon isotope (see the composition shown in Table 7 below), and then the E. coli cell samples and acetic acid samples contained in the culture solution were analyzed. Analysis of the mass number of amino acid constituting E. coli using Escherichia coli cell samples was carried out using gas-chromatography/mass spectroscopy after hydrolysis of all the proteins constituting Escherichia coli under strongly acidic and high-temperature conditions (Zamboni et al., Nat. protocols, 4:6, 878-892, 2009). Analysis of the acetic acid sample contained in the culture solution was carried out by lyophilizing the culture solution, separating the acetic acid contained in the culture solution into acetate, and conducting analysis using the gas-chromatography/mass-spectroscopy analysis method.
The assimilation of formic acid in the cyclic formic acid and carbon dioxide assimilation pathway developed in the present invention is carried out though the bonding between tetrahydrofolate and formic acid among the two cyclic metabolic pathways, as shown in
As shown in
Meanwhile, the results of quantitative analysis of formic acid assimilation of the wild-type control (DH5α WT) and recombinant E. coli (DH5α THF) and comparison of assimilation efficiency showed that recombinant E. coli (DH5α THF) used formic acid as a main carbon source, while the wild-type control (DH5α WT) used almost no formic acid (
From the above results, it could be seen that the recombinant E. coli according to the present invention exhibited significantly improved assimilation efficiency compared to that of the wild-type control.
Whether or not the formic acid assimilated through the cyclic formic acid and carbon dioxide assimilation metabolic pathway developed in the present invention was assimilated into the central carbon metabolism (CCM) of E. coli. was verified. The formic-acid-derived carbon assimilated through the metabolic pathway of Example 2-1 was migrated to 3-hydroxypyruvate through serine-glyoxylate transaminase and corresponded to the carbon at position 3 of pyruvate by a series of reactions. At this time, the proportion of carbon isotopes present in pyruvate could be determined by analysis of alanine and valine (Zelcbuch et al., Biochemistry, vol. 55:17, 2423-2426, 2016). Based on this principle, the carbon isotopes contained in pyruvate were analyzed. As a result, as shown in
The identification of the production of acetyl-CoA and regeneration of glyoxylate through the carbon dioxide assimilation process, which is the last part of the cyclic process of the cyclic formic acid and carbon dioxide assimilation pathway developed in the present invention, was carried out by measuring the contents of carbon isotopes in acetic acid produced by the recombinant E. coli of Example 1 and the wild-type E. coli. That is, the carbon-dioxide-derived carbon corresponds to the carbon at position 1 of the acetyl-CoA synthesized by the cyclic formic acid and the carbon dioxide assimilation pathway developed in the present invention. Generally, acetyl-CoA is converted at a certain proportion into acetic acid in Escherichia coli. Therefore, analysis of acetic acid provides analysis of the content of isotopes in acetyl-CoA.
The results of analysis of the carbon isotope content in acetyl-CoA based on this principle showed that the recombinant Escherichia coli (DH5α ST3) having the metabolic pathway had an increased isotope content in acetic acid compared to wild-type Escherichia coli. Therefore, it could be seen that the recombinant E. coli according to the present invention was effective for carbon dioxide assimilation and glyoxylate regeneration.
The pyruvate formate lyase was further introduced into the cyclic metabolic pathway to identify whether or not pyruvate was synthesized from acetyl-CoA and formic acid through the reverse reaction of the enzyme, which can be identified through carbon isotope analysis. That is, when formic acid and carbon dioxide labeled with a carbon isotope having a mass number of 13 are supplied, pyruvate synthesized through the corresponding cyclic metabolic pathway and the reverse reaction of pyruvate formate lyase has formic-acid-derived carbon at positions 1 and 3 and carbon-dioxide-derived carbon at position 2.
Based on this principle, pyruvate was analyzed by the same method as the carbon isotope analysis used in Example 2-2. As a result, it could be seen that, in the case of recombinant E. coli (DH5α ST2) having a cyclic metabolic pathway produced in the present invention, the proportion of amino acids increased by 3 or more above the original mass number of corresponding amino acids in valine and alanine, was significantly increased compared to wild-type E. coli.
Carbon isotope analysis was performed to verify whether or not the recombinant E. coli produced in Example 1 could assimilate formic acid and carbon dioxide to produce glycine, serine and pyruvate. For the verification of formic acid assimilation, the experiments were carried out using the wild-type Escherichia coli (DH5α WT), as a control group, and recombinant E. coli (DH5α RG2) introduced with formate-tetrahydrofolate ligase encoded by a nucleic acid molecule represented by SEQ ID NO: 7, methenyl tetrahydrofolate cyclohydrolase encoded by the nucleic acid molecule represented by SEQ ID NO: 8, methylene-tetrahydrofolate dehydrogenase encoded by the nucleic acid molecule represented by SEQ ID NO: 9, and a glycine cleavage complex encoded by the nucleic acid molecule represented by SEQ ID NO: 63 among the recombinant Escherichia coli produced in Example 1.
In order to verify the improvement in assimilation efficiency of formic acid and carbon dioxide, experiments were performed using recombinant Escherichia coli (DH5α RG4) obtained by deleting a gcvR gene (SEQ ID NO: 66, NCBI information: NC_000913.3, Region 2599906-2600478) and changing a promoter of a gene constituting the glycine cleavage complex {substituting SEQ ID NO: 67 (NCBI information: NC_000913.3 Region 3049125-3050667) with SEQ ID NO: 68) in the recombinant E. coli (DH5α RG2).
For the carbon isotope analysis, the control and experimental E. coli were cultured in M9 medium containing formate and bicarbonate ion labeled with 13C carbon isotope (see the composition shown in Table 7 above), and then the E. coli cell samples were analyzed. Analysis of the mass number of amino acid constituting E. coli using E. coli cell samples was carried out using gas-chromatography/mass spectroscopy after hydrolysis of all of the proteins constituting Escherichia coli under strongly acidic and high-temperature conditions (Zamboni et al., Nat. protocols, 4:6, 878-892, 2009).
The recombinant E. coli (DH5α RG2) synthesized glycine from one molecule of formic acid and one molecule of carbon dioxide through the metabolic pathway shown in
In order to improve the synthesis of glycine, serine and pyruvate from formic acid, a recombinant strain (DH5α RG4) was prepared by changing a regulatory gene (gcvR) controlling the expression of the glycine cleavage complex and the promoter of the glycine cleavage complex in the recombinant E. coli (DH5α RG2). The synthesis of glycine, serine and pyruvate from formic acid and carbon dioxide was analyzed. As a result, it could be seen that 96%, 86% and 7.3% of glycine, serine and pyruvate, respectively, were synthesized from formic acid and carbon dioxide.
In conclusion, based on the examples described above, the recombinant E. coli according to the present invention effectively acts on assimilation of formic acid and carbon dioxide through the cyclic metabolic pathway identified in the present invention, and further introduction of pyruvate formate lyase offers a significant increase in pyruvate synthesis efficiency. In addition, it can be seen that glycine, serine and pyruvate can be synthesized with high efficiency from formic acid and carbon dioxide through formic acid and carbon dioxide assimilation metabolic pathway.
Although specific configurations of the present invention have been described in detail, those skilled in the art will appreciate that this detailed description is provided as preferred embodiments for illustrative purposes and should not be construed as limiting the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the accompanying claims filed and equivalents thereto.
Synthetic promoter 100: BBa23100 synthetic promoter
ftfL: SEQ ID NO: 7, formate-tetrahydrofolate ligase
fch: SEQ ID NO: 8, methenyl tetrahydrofolate cyclohydrolase
mtdA: SEQ ID NO: 9, methylene-tetrahydrofolate dehydrogenase
rrnBT: rrnB terminator
APr: Ampicillin resistance gene
pBR322 origin: pBR322 replication origin
mtkA(CM4): SEQ ID NO: 3, malate-CoA ligase β-subunit
mtkB(CM4): SEQ ID NO: 4, malate-CoA ligase α-subunit
mcl: SEQ ID NO: 5, malyl-CoA lyase
sga: SEQ ID NO: 6, serine-glyoxylate aminotransferase
CmR: chloramphenicol resistance gene
p15A: p15A replication origin
P23100: BBa23100 synthetic promoter
sgaAT: SEQ ID NO: 21, serine-glyoxylate aminotransferase
rrnBT1T2: rrnB terminator
sucC2 OP: SEQ ID NO: 17, succinyl-CoA:(S)-malate CoA-transferase
sucD2 OP: SEQ ID NO: 18, succinyl-CoA:(S)-malate CoA-transferase
gcvT: SEQ ID NO: 63, T subunit among glycine cleavage complex
gcvH: SEQ ID NO: 63, H subunit among glycine cleavage complex
gcvP: SEQ ID NO: 63, P subunit among glycine cleavage complex
trc-pro: Trc promoter
LacIq: mutant lad repressor
The present invention provides a novel microorganism introduced with a novel cyclic metabolic pathway through which C3 or higher organic carbon compounds can be synthesized from formic acid and carbon dioxide, thereby effectively synthesizing pyruvate as a C3 organic compound using carbon dioxide, which is abundant in nature, and formic acid, which is of low toxicity and suitable for assimilation (anabolic) reactions in view of reaction kinetics and which can be easily and rapidly synthesized from carbon dioxide. As a result, the present invention is effective in economically producing organic carbon compounds, reducing carbon dioxide, which is the main cause of global warming, and synthesizing various high-value-added compounds from pyruvate as an intermediate product.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2016-0180223 | Dec 2016 | KR | national |
| 10-2017-0151642 | Nov 2017 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2017/013171 | 11/20/2017 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/124475 | 7/5/2018 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20030124687 | Gunji et al. | Jul 2003 | A1 |
| 20130196359 | Siegel et al. | Aug 2013 | A1 |
| 20150218528 | Bar-Even | Aug 2015 | A1 |
| Number | Date | Country |
|---|---|---|
| 102007048625 | Apr 2009 | DE |
| 2014-155455 | Aug 2014 | JP |
| 100679638 | Jan 2007 | KR |
| 1020090117739 | Nov 2009 | KR |
| 2008101857 | Aug 2008 | WO |
| 2009062190 | May 2009 | WO |
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| 20190338316 A1 | Nov 2019 | US |
