Recombinant microorganism with improved formic acid and carbon dioxide assimilation efficiency and method for producing useful substances from formic acid and carbon dioxide by using same

Abstract

The present invention relates to: a recombinant microorganism with increased formic acid and carbon dioxide assimilation efficiency through foreign gene introduction or amplification and genome manipulation; and a method for producing useful substances from formic acid and carbon dioxide by using same. Specifically, the present invention relates to: a recombinant microorganism with enhanced carbon dioxide and formic acid assimilation through the introduction or amplification and genome manipulation of genes that can be involved in formic acid and carbon dioxide assimilation pathways; and a method for producing useful substances by using the recombinant microorganism, in which formic acid and carbon dioxide are used alone as carbon sources to improve growth.

Description

Recombinant microorganism with improved assimilation efficiency of formic acid and carbon dioxide and a method for producing useful substances from formic acid and carbon dioxide using the same

[0001] The present invention relates to a recombinant microorganism in which the assimilation efficiency of formic acid and carbon dioxide is increased through the introduction or amplification of foreign genes and genomic manipulation, and a method for producing useful substances from formic acid and carbon dioxide using the same. Specifically, the invention relates to a recombinant microorganism in which the assimilation of carbon dioxide and formic acid is enhanced through the introduction or amplification of genes capable of participating in the formic acid and carbon dioxide assimilation pathways and genomic manipulation, and a method for producing useful substances by improving growth using said recombinant microorganism as a carbon source solely of formic acid and carbon dioxide.

[0002]

[0003] The increase in carbon dioxide caused by the use of fossil fuels is one of the primary causes of climate change worldwide. Consequently, methods for producing chemicals in a sustainable manner are gaining attention. In particular, research into converting greenhouse gases, such as carbon dioxide, into useful materials is crucial for reducing environmental burdens and fostering sustainable industries. Research on the conversion of single-carbon compounds (C1 compounds), such as carbon dioxide, can be broadly categorized into chemical and biological conversion. Chemical conversion proceeds through electrochemical reactions utilizing metal and non-metal catalysts. While these chemical reactions proceed at a relatively rapid rate and offer high conversion rates and scalability, they are limited by the low diversity of products. In the case of biological conversion, C1 compounds are converted using natural C1 metabolic strains or artificial C1 metabolic pathways. This biological C1 conversion process has the advantage of generating environmentally less harmful byproducts and producing a diverse range of products. However, it is essential to supply the reducing power required by cells during the process of assimilating biological C1 compounds.

[0004] Formic acid is a C1 compound capable of electrochemically converting carbon dioxide with high efficiency. Since formic acid can exist in a liquid state at room temperature, it is easy to store and transport. Furthermore, formic acid can be utilized by formic acid dehydrogenase (Fdh) as the reducing power required for carbon source assimilation in cells. Due to these advantages, formic acid is attracting attention as a carbon source for microbial metabolism. In prior research, the inventors designed and verified a novel metabolic pathway to convert formic acid, a C1 compound, into useful compounds composed of multiple carbon atoms (Korean Registered Patent No. 10-2000755). Additionally, by introducing and enhancing a metabolic pathway to synthesize pyruvate from carbon dioxide and formic acid, they constructed a recombinant microorganism capable of growing solely from carbon dioxide and formic acid (Korean Registered Patent No. 2690858).

[0005] However, conventionally constructed recombinant microorganisms exhibit low growth rates and maximum cell densities under conditions where only formic acid and carbon dioxide are provided as carbon sources. This limits the development of efficient C1 compound conversion processes.

[0006] Against this technical background, the inventors of the present application developed a recombinant microorganism that increases assimilation efficiency by using only carbon dioxide and formic acid as carbon sources by introducing or amplifying genes involved in the assimilation pathways of formic acid and carbon dioxide; confirmed that higher growth rates and cell densities can be achieved through a growth-optimized medium and culture method utilizing such microorganism; and completed the present invention.

[0007]

[0008] Summary of the Invention

[0009] The objective of the present invention is to provide a recombinant microorganism with increased assimilation ability of formic acid and carbon dioxide.

[0010] Another objective of the present invention is to provide a method for producing a useful substance through a culture medium and a culture method that can efficiently grow the recombinant microorganism from formic acid and carbon dioxide using the above-mentioned recombinant microorganism.

[0011] To achieve the above objective, the present invention provides a recombinant microorganism with enhanced carbon dioxide and formic acid assimilation, wherein a gene encoding alanine-glyoxylate aminotransferase 1; and / or a gene encoding L-serine ammonia-lyase are introduced or amplified into a host microorganism having a central carbon assimilation circuit.

[0012] The present invention also provides a method for producing a useful substance, comprising: (a) culturing the recombinant microorganism using formic acid and carbon dioxide as carbon sources to produce a useful substance; and (b) recovering the produced useful substance.

[0013]

[0014] Figure 1 illustrates the key metabolic pathways in the carbon dioxide and formic acid assimilation processes of recombinant microorganisms, as well as the genes, enzymes, and metabolites involved therein.

[0015] Figure 2 illustrates a plasmid containing genes involved in carbon dioxide and formic acid assimilation, a formic acid dehydrogenase derived from the genus Candida, a variant of formic acid dehydrogenase derived from the genus Arabidopsis, alanine-glyoxylate aminotransferase 1 derived from the genus Saccharomyces, and L-serine ammonia-lyase derived from the genus Cupriavidus.

[0016] Figure 3 is a graph showing cell growth measured when cultured with only carbon dioxide and formic acid according to the strains of FC8 and FC9.

[0017] Figure 4 is a graph showing cell growth and formic acid concentration in the medium when cultured in a flask with only carbon dioxide and formic acid according to the FC9 and FC10 strains.

[0018] Figure 5 is a graph showing cell growth and formic acid concentration in the medium when cultured in a flask with only carbon dioxide and formic acid according to the FC9 and FC11 strains.

[0019] Figure 6 is a graph showing cell growth and formic acid concentration in the medium when cultured in a flask with only carbon dioxide and formic acid, depending on the FC9 and FC12 strains.

[0020] Figure 7 is a graph showing the growth and formic acid concentration in the culture medium according to conditions in which sodium selenite at concentrations of 0.2 μM, 1 μM, 2 μM, and 20 μM was added to a culture medium that provides only formic acid and carbon dioxide as carbon sources.

[0021] Figure 8 is a graph measuring cell growth according to the optimization of the culture method of the FC9 strain.

[0022]

[0023] Specific details for implementing the invention

[0024] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by a skilled expert in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0025] Coenzyme-mediated redox cofactors play a crucial role in cellular metabolism. These cofactors exist primarily in the form of NADH, NADPH, and FADH2 and are involved in electron transfer and energy production across various metabolic pathways. These coenzymes catalyze the interconversion of cofactors via transhydrogenases, which are redox regulatory enzymes, thereby providing metabolic flexibility between the catabolism of NADPH formation and the anabolism or stress-based consumption of NADPH.

[0026] In addition, mutations in the corresponding gene were observed in the laboratory adaptive evolution process in prior research literature (A quantitative method for proteome reallocation using minimal regulatory interventions. Nat Chem Biol 16, 1277 (2020).).

[0027] Through this, it was confirmed that the growth rate of E. coli increased under culture conditions using only formic acid and carbon dioxide by enhancing expression through the replacement of the promoter of the gene encoding pyridine nucleotide transhydrogenase present in the E. coli genome.

[0028] Formic acid is a C1 compound capable of electrochemically converting carbon dioxide with high efficiency. Since formic acid can exist in a liquid state at room temperature, it is easy to store and transport. Furthermore, formic acid can be utilized by formic acid dehydrogenase (Fdh) as the reducing power required for carbon source assimilation in cells. Due to these advantages, formic acid is attracting attention as a carbon source for microbial metabolism. In prior research, the inventors designed and verified a novel metabolic pathway to convert formic acid, a C1 compound, into useful compounds composed of multiple carbon atoms (Korean Registered Patent No. 10-2000755). Additionally, by introducing and enhancing a metabolic pathway to synthesize pyruvate from carbon dioxide and formic acid, they constructed a recombinant microorganism capable of growing solely from carbon dioxide and formic acid (Korean Registered Patent No. 2690858).

[0029] Reflecting this, alanine-glyoxylate aminotransferase 1 from Saccharomyces yeast, which can be involved in the formic acid and carbon dioxide assimilation pathways, was introduced to introduce the glycine metabolic pathway, and L-serine ammonia-lyase from Cupriavidus was introduced to enhance the conversion of assimilated serine to pyruvate. In addition, the efficiency of formic acid and carbon dioxide assimilation was increased by removing the glucose phosphotransferase system constituent protein (ptsG), which can be involved in inhibiting carbon catabolism.

[0030] In addition, the present invention was completed by developing a culture medium supplemented with trace elements and antifoaming agents, and by developing a culture method that improves the supply of carbon dioxide and nitrogen sources, thereby confirming that the cell growth rate and cell density can be increased under culture conditions that provide only formic acid and carbon dioxide as carbon sources.

[0031] In the present invention, a recombinant microorganism capable of growing using only carbon dioxide and formic acid (Korean Registered Patent No. 2690858) was further improved through metabolic engineering, and by improving the culture medium and culture method together, it was confirmed that the growth rate can be increased using only formic acid and carbon dioxide.

[0032] Accordingly, in one aspect, the present invention relates to a recombinant microorganism in which carbon dioxide and formic acid assimilation are enhanced, wherein a gene encoding alanine-glyoxylate aminotransferase 1; and / or a gene encoding L-serine ammonia-lyase are introduced or amplified into a host microorganism having a central carbon assimilation circuit.

[0033] The above central carbon assimilation pathway may refer to a metabolic pathway applied to the assimilation process of a central carbon, for example, formic acid and / or carbon dioxide.

[0034] Figure 1 schematically illustrates the core metabolic pathways in the carbon dioxide and formic acid assimilation processes of recombinant microorganisms, as well as the genes, enzymes, and metabolites involved therein. In the present invention, the formic acid assimilation cycle is a cyclic circuit in which carbon dioxide and formic acid are assimilated into pyruvate composed of three carbon atoms in microorganisms. Figure 1 illustrates the genes, coenzymes, and energy transfer substances involved in the formic acid assimilation cycle of E. coli.

[0035] In a host microorganism having formic acid and carbon dioxide assimilation circuits, a recombinant microorganism was constructed by enhancing the expression of the redox regulatory enzyme pyridine nucleotide transhydrogenase gene, removing the gene encoding a protein of the glucose phosphotransferase system, and introducing the genes for alanine-glyoxylate aminotransferase and L-serine ammonia decomposition enzyme involved in glycine supply.

[0036] In the present invention, the formic acid assimilation circuit can synthesize carbon compounds of C3 or higher by combining with the central carbon assimilation circuit, and the host microorganism has the central carbon assimilation circuit by i) having the central carbon assimilation circuit internally; or ii) having the central carbon assimilation circuit introduced externally.

[0037] The gene encoding alanine-glyoxylate aminotransferase 1 involved in the above glycine metabolism may be an alanine-glyoxylate aminotransferase 1 coding gene of Saccharomyces yeast that may be involved in the formic acid and carbon dioxide assimilation pathways.

[0038] The gene encoding the alanine-glyoxylate aminotransferase 1 may be characterized by including, for example, the sequence of SEQ ID NO. 1.

[0039] The gene coating the L-serine ammonia-lyase involved in the above serine metabolism can enhance the conversion of assimilated serine to pyruvate by introducing L-serine ammonia-lyase derived from the genus Cupriavidus.

[0040] The gene coating the above L-serine ammonia degrading enzyme may be characterized by including, for example, the sequence of SEQ ID NO. 2.

[0041] In the present invention, the recombinant microorganism has the expression of the gene encoding pyridine nucleotide transhydrogenase of the host cell further enhanced.

[0042] In the present invention, the recombinant microorganism has the gene (ptsG) encoding pyridine nucleotide transhydrogenase of the host cell removed.

[0043] Specifically, the present invention may be characterized in that a gene coding for pyridine nucleotide transhydrogenase is overexpressed and a gene coding for a glucose phosphotransferase system constituent protein (ptsG) is deleted.

[0044] The gene encoding the pyridine nucleotide transhydrogenase may be the pntA or pntB gene. The gene encoding the pyridine nucleotide transhydrogenase may be the pntA gene containing the sequence of SEQ ID NO. 3 or the pntB gene containing the sequence of SEQ ID NO. 4.

[0045] The gene encoding the above pyridine nucleotide transhydrogenase may be overexpressed by any one strong promoter selected from the group consisting of the trc promoter, tac promoter, T7 promoter, lac promoter, and trp promoter.

[0046] The gene encoding the above-mentioned pyridine nucleotide transhydrogenase may be characterized by having its expression enhanced by replacing the intrinsic promoter with any one strong promoter selected from the group consisting of trc promoter, tac promoter, T7 promoter, lac promoter, and trp promoter, and may be characterized by being overexpressed through a plasmid overexpression system.

[0047] The above glucose phosphotransferase system constituent protein coding gene may include the sequence of SEQ ID NO. 9.

[0048]

[0049] It may additionally include one or more genes encoding for selected from the group consisting of formate-tetrahydrofolate ligase, methenyl tetrahydrofolate cyclohydrolase, methylene-tetrahydrofolate dehydrogenase, formate dehydrogenase, and formate dehydrogenase mutant.

[0050] The formate-tetrahydrophorate ligase coding gene comprises the sequence of SEQ ID NO. 10; the methylene-tetrahydrophorate cyclohydrolase coding gene comprises the sequence of SEQ ID NO. 11; the methylene-tetrahydrophorate dehydrogenase comprises the sequence of SEQ ID NO. 12; the formic acid dehydrogenase comprises the sequence of SEQ ID NO. 13; or the formic acid dehydrogenase variant may comprise the sequence of SEQ ID NO. 14.

[0051]

[0052]

[0053]

[0054] A gene encoding one or more selected from the group consisting of formate-tetrahydrofolate ligase, methenyl tetrahydrofolate cyclohydrolase, methylene-tetrahydrofolate dehydrogenase, formate dehydrogenase, and formate dehydrogenase mutant may be characterized by being cloned and introduced into a vector containing a replication origin having copy numbers 1 to 12, and preferably may be characterized by being cloned and introduced into a vector containing a replication origin having copy numbers 1 to 5, but is not limited thereto.

[0055] The above host microorganism may be characterized as being selected from the group consisting of the genera Escherichia, Manheimia, Rhodobacter, and Methylobacterium, but is not limited thereto.

[0056] The recombinant microorganism according to the present invention may be characterized by increased assimilation efficiency of formic acid and carbon dioxide by utilizing only formic acid and carbon dioxide as carbon sources.

[0057] The gene of the present invention may undergo various modifications to the coding region within a range that does not alter the amino acid sequence of the protein expressed from the coding region, and may also undergo various modifications or alterations to parts excluding the coding region within a range that does not affect gene expression, and such modified genes are also included within the scope of the present invention.

[0058] Accordingly, the present invention also comprises a polynucleotide having a base sequence substantially identical to that of the said gene and a fragment of said gene. A substantially identical polynucleotide means a gene encoding an enzyme having the same function as that used in the present invention, regardless of sequence homology. A fragment of said 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.

[0059] Furthermore, the amino acid sequence of the protein, which is the expression product of the gene of the present invention, can be obtained from various biological resources, such as microorganisms, within a range that does not affect the potency and activity of the corresponding enzyme, and proteins obtained from such other biological resources are also included within the scope of the present invention.

[0060] Accordingly, the present invention also comprises a polypeptide having a substantially identical amino acid sequence to the protein and a fragment of said polypeptide. A substantially identical polypeptide means a protein having the same function as that used in the present invention, regardless of homology of the amino acid sequence. A fragment of said polypeptide also means a protein having the same function as that used in the present invention, regardless of the length of the fragment.

[0061] In the present invention, "vector" refers to a DNA product containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA within a suitable host. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. When transformed into a suitable host, the vector may replicate and function independently of the host genome, or in some cases, be incorporated into the genome itself. Since plasmids are the most commonly used form of vector currently, "plasmid" and "vector" are sometimes used interchangeably in the specification of the present invention. For the purposes of the present invention, it is preferable to use plasmid vectors. A typical plasmid vector that can be used for this purpose has a structure comprising (a) a replication initiation site that enables efficient replication to include several to hundreds of plasmid vectors per host cell, (b) an antibiotic resistance gene that enables the selection of host cells transformed with the plasmid vector, and (c) a restriction enzyme cleavage site into which foreign DNA fragments can be inserted. Even if a suitable restriction enzyme cleavage site is not present, the vector and foreign DNA can be easily ligated using synthetic oligonucleotide adapters or linkers according to conventional methods. After ligation, the vector must be transformed into a suitable host cell. Transformation can be easily achieved using the calcium chloride method or electroporation (Neumann, et al, EMBO J, 1:841, 1982).

[0062] In the present invention, the meaning of gene weakening is that the expression of the gene is relatively lower compared to the wild type, or the function or activity of the protein encoded by the gene is reduced compared to the wild type.

[0063] The vector used for enhancing or overexpressing a gene according to the present invention may be an expression vector known in the art.

[0064] A base sequence is "operably linked" when positioned in a functional relationship with another nucleic acid sequence. This may be a gene and a regulatory sequence(s) linked in such a way that gene expression is enabled when an appropriate molecule (e.g., a transcription-activating protein) binds to the regulatory sequence(s). For example, DNA for a pre-sequence or secretion leader is operably linked to DNA for a polypeptide when expressed as a pre-sequence protein participating in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence when it influences the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence when it influences the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence when positioned to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are in contact, and in the case of a secretion leader, they are in contact and exist within the reading frame. However, an enhancer does not need to be in contact. The linking of these sequences is a convenient restriction This is performed by ligation at the enzyme site. If such a site does not exist, a synthetic oligonucleotide adaptor or linker is used according to conventional methods.

[0065] As is well known in the art, in order to increase the expression level of a transformed gene in a host cell, the gene must be operably linked to transcriptional and translational expression regulatory sequences that are functional within the selected expression host. Preferably, the expression regulatory sequences and the gene are contained within a single recombinant vector that includes both a bacterial selection marker and a replication origin. If the host cell is a eukaryotic cell, the recombinant vector must additionally include expression markers useful within the eukaryotic expression host.

[0066] Host cells transformed by the aforementioned recombinant vector constitute another aspect of the present invention. As used herein, the term "transformation" means the introduction of DNA into a host so that the DNA becomes replicable as an extrachromosomal factor or through the completion of chromosomal integration.

[0067] Of course, it must be understood that not all vectors function equally in expressing the DNA sequence of the present invention. Likewise, not all hosts function equally for the same expression system. However, those skilled in the art can make appropriate selections from among various vectors, expression regulatory sequences, and hosts without departing from the scope of the present invention and without excessive experimental burden. For example, when selecting a vector, the host must be considered, as the vector must be replicated within it. The copy number of the vector, the ability to regulate the copy number, and the expression of other proteins encoded by said vector, such as antibiotic markers, must also be considered.

[0068] Furthermore, the gene introduced in the present invention may be characterized by being introduced into the genome of a host cell and existing as a chromosomal factor. It will be obvious to those skilled in the art to which the present invention pertains that inserting the said gene into the genomic chromosome of a host cell will produce the same effect as when a recombinant vector is introduced into the host cell as described above.

[0069] The above recombinant microorganism may be capable of biosynthesizing pyruvate, glycine, or serine assimilated from formic acid and carbon dioxide.

[0070] The above host microorganism may be selected from the group consisting of, for example, Escherichia, Mannheimia, Rhodobacter, and Methylobacterium, but is not limited thereto.

[0071] Recombinant E. coli was produced by transforming E. coli with the constructed recombinant plasmid. The E. coli used in this invention was E. coli DH5α (Invitrogen, USA), and the transformation into E. coli was performed using a chemical transformation method commonly used in the industry.

[0072] Meanwhile, in the present invention, it was confirmed that by adding sodium selenite and an antifoaming agent to the culture medium of the recombinant microorganism under formic acid and carbon dioxide conditions, and controlling the air and carbon dioxide supply rates, the culture time of the recombinant microorganism could be shortened by 2.27 times and the maximum cell density could be increased by 2.26 times.

[0073] Accordingly, the present invention relates, in another aspect, to a method for producing a useful substance comprising the following steps:

[0074] (a) a step of producing a useful substance by culturing the recombinant microorganism using formic acid and carbon dioxide as carbon sources; and (b) a step of recovering the produced useful substance.

[0075] The above useful substances can be produced, for example, using a C3 compound as an intermediate. Among the above recombinant microorganisms, glycine, serine, and the C3 compound pyruvate can be synthesized from formic acid and carbon dioxide.

[0076] The above useful substances are butanol, isobutanol, 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-isobutanol, putrescine, L-ornithine, arginine, polycyclic aromatic hydrocarbon (PHA), polylactate, polylactate-co-glycolate, polyisovalerate, polyhydroxybutyrate (PHB), 4-hydroxybutyrate, biodiesel, gasoline, olefin, 5-aminovaleric acid, gamma-aminobutyric acid, 3-hydroxyspiropionic acid, 3-aminopropionic acid, acrylic acid, 1,3-diaminopropane, caprolactam, threonine, It may be characterized by being selected from the group consisting of valine, isoleucine, fumaric acid, malic acid, succinic acid, ceramide, astaxanthin, silibinin, lycopene, lutein, and retinol, but is not limited thereto.

[0077] Step (a) above may be characterized by culturing the recombinant microorganism in a culture medium supplemented with 0.2 μM to 20 μM of sodium selenite.

[0078] In the present invention, the culture medium may be characterized by containing sodium selenite at a concentration of 0.2 μM to 20 μM, but is not limited thereto. Additionally, it may be characterized by containing the antifoaming agent antifoam 204 at a concentration of 0.01%, but is not limited thereto.

[0079] In step (a) above, formic acid may be maintained at a concentration of 2 to 3 g / l and the pH may be maintained at 6.6 to 7.0, and formic acid and ammonium chloride may be supplied. The ammonium chloride may be added at a concentration of 2 g / L or 20 g / L to the formic acid supply solution during culture, but is not limited thereto.

[0080] In addition, in step (a) above, the initial air and carbon dioxide supply amounts can be increased from 450 cc / min and 50 cc / min, respectively, to 900 cc / min and 100 cc / min, respectively, and culture can be performed. The present invention may be characterized by increasing the air and carbon dioxide supply amounts from an initial 450 cc / min and 50 cc / min to 900 cc / min and 100 cc / min, respectively, but is not limited thereto.

[0081] The recombinant microorganism according to the present invention can grow at a faster growth rate and higher cell density using only carbon dioxide and formic acid. The recombinant microorganisms previously developed by the inventors were capable of growing using only carbon dioxide and formic acid without glucose supply, but their growth rate and peak cell density were low. However, the recombinant microorganism according to the present invention increased the growth rate and peak cell density using only carbon dioxide and formic acid by further introducing a plasmid containing a gene encoding alanine-glyoxylate aminotransferase, which is involved in glycine supply, to the recombinant microorganism capable of growing using only carbon dioxide and formic acid, thereby enhancing the gene expression of pyridine nucleotide transhydrogenase, a redox regulatory enzyme.

[0082] In the present invention, culture and fermentation conditions capable of maximizing the growth of the recombinant microorganism were developed. This is based on the previously filed E. coli growing only with carbon dioxide and formic acid, which showed a maximum cell density of OD600 7.38 during a culture time of 242.3 hours and a maximum cell density of OD600 27.7 during a culture time of 450 hours. This was achieved by adding sodium selenite and an antifoaming agent to the medium, controlling the supply of air and carbon dioxide, and supplying ammonium chloride as a nitrogen source.

[0083]

[0084] Examples

[0085] The present invention will be explained in more detail below through examples. These examples are merely for the purpose of explaining the present invention more specifically, and it will be obvious to those skilled in the art that the technical scope of the present invention is not limited by these examples.

[0086] Example 1: Development of a recombinant microorganism with increased carbon dioxide and formic acid assimilation efficiency through metabolic engineering strain improvement

[0087] To develop a strain with increased assimilation efficiency using only carbon dioxide and formic acid, the gene expression encoding pyridine nucleotide transhydrogenase of Escherichia coli was enhanced, the gene (ptsG) encoding a glucose phosphotransferase system constituent protein was removed, and a recombinant microorganism was developed by introducing genes encoding alanine-glyoxylate aminotransferase 1 derived from the genus Saccharomyces and L-serine ammonia-lyase derived from the genus Cupriavidus.

[0088] Specifically, among the foreign gene fragments required for plasmid production, the alanine-glyoxylate aminotransferase gene was synthesized in the form of a gene fragment by optimizing it into an E. coli gene codon form, and the L-serine ammonia degradase was prepared by using the genomic DNA of a microorganism possessing the gene as a template, performing PCR using primers designed for gene amplification, and then recovering and purifying the amplified gene fragment. The sequences of the gene and primers are shown below.

[0089]

[0090]

[0091]

[0092] The plasmid backbone gene fragment was prepared by performing PCR using the primers mentioned above, and then recovering and purifying the amplified gene fragment.

[0093] Plasmids were constructed using the Gibson assembly method (Gibson et al., Nat. methods, 6:5, 343-345, 2009), which is commonly used for assembling gene fragments, and each plasmid was constructed to contain one or more of the foreign genes listed in the table above. (Fig. 2)

[0094]

[0095] Recombinant E. coli was produced by transforming E. coli with the recombinant plasmid produced by the above method. The E. coli used in this invention was E. coli DH5α (Invitrogen, USA), and the transformation into E. coli was performed using a chemical transformation method commonly used in the industry.

[0096] Here, gene expression encoding pyridine nucleotide transhydrogenase was enhanced. The enhanced genes are as follows.

[0097]

[0098]

[0099]

[0100] The following strains were produced by introducing the p518FA1R_agx1sce and p518FA1R_sdaAcup plasmids into the strain produced above.

[0101]

[0102] To confirm the improvement in the carbon dioxide and formic acid assimilation capabilities of the synthesized recombinant E. coli, flask cultures were first performed in formic acid and carbon dioxide culture media, with the aforementioned recombinant E. coli serving as the experimental group and the previously established E. coli as the control group.

[0103]

[0104] To confirm the enhancement of carbon dioxide and formic acid assimilation capabilities of the synthesized recombinant E. coli, the recombinant E. coli were first used as the experimental group and the previously constructed E. coli as the control. To verify the growth rate and peak cell density using only carbon dioxide and formic acid induced by enhanced pyridine nucleotide transhydrogenase gene expression in the AKOpntABPC strain, bioreactor culture of the strain was performed using the FC8 strain as the control. Fed-batch fermentation was carried out in M9 medium (Table 7) while maintaining an FA concentration of 3 g / L. The FC9 strain grew from an OD600 of 1.01 to 10.2 over 206 hours, showing a 2.5-fold improvement in growth rate compared to the cell density of the control FC8 strain (OD600 of 4.08) at the same time (Fig. 3).

[0105] In addition, to confirm the growth rate and peak cell density using only carbon dioxide and formic acid following the introduction of the alanine-glyoxylate aminotransferase 1 coding gene derived from the genus Saccharomyces via the above FC10, flask culture of the FC10 strain was performed in a carbon dioxide-supplying culture vessel using the FC9 strain as a control. Under flask culture conditions, the FC10 strain grew from an OD600 of 0.134 to a peak cell density of OD600 of 2.85 over a culture period of 288 hours, showing a 1.61-fold improvement in growth rate compared to the peak cell density of the control FC9 strain (OD600 of 1.76) over the same period, and exhibited a faster rate of formic acid consumption (Fig. 4).

[0106] In addition, to confirm the growth rate and peak cell density using only carbon dioxide and formic acid by introducing L-serine ammonia-lyase derived from the genus Cupriavidus via the above FC11, flask culture of the above FC11 strain was carried out in a carbon dioxide-supplying culture medium using the FC9 strain as a control. Under flask culture conditions, the FC10 strain grew from an OD600 of 0.582 to a peak cell density of OD600 of 4.70 over a culture period of 200 hours, showing a 1.20-fold improvement in growth rate compared to the peak cell density of the control FC9 strain (OD600 of 3.91) over the same period, and exhibited a faster rate of formic acid consumption (Fig. 5).

[0107] In addition, to determine the growth rate and peak cell density of the above FC12 using only carbon dioxide and formic acid by removing the glucose phosphotransferase system constituent protein (ptsG), flask culture of the above FC11 strain was carried out in a carbon dioxide-supplying culture vessel using the FC9 strain as a control. Under flask culture conditions, the FC10 strain grew from an OD600 of 0.577 to a peak cell density of OD600 of 4.51 over a culture time of 200 hours, showing a 1.33-fold improvement in growth rate compared to the peak cell density of the control FC9 strain (OD600 of 3.37) over the same period, and exhibited a faster rate of formic acid consumption (Fig. 6).

[0108] Flask culture was performed in acid and carbon dioxide culture media.

[0109]

[0110] Example 2: Development of a culture medium and culture method characterized by efficient growth when only formic acid and carbon dioxide are provided as carbon sources

[0111] A culture medium optimized for growth using only formic acid and carbon dioxide was developed using the above strain, thereby improving the growth rate and peak cell density of the FC9 strain. The optimal concentration of sodium selenate was determined by performing flask cultures with sodium selenate added to the culture medium indicated in Table 7 at concentrations ranging from 0.2 μM to 20 μM. In the culture medium with 0.2 μM of sodium selenate added, the strain grew from an OD600 of 0.535 to a peak cell density of OD600 of 2.22 over 166 hours of culture, showing a 1.19-fold improvement in growth rate and increased formic acid consumption compared to the peak cell density of OD600 of 1.87 in the control group without added sodium selenate (Fig. 7).

[0112] In addition, a culture method optimized for growth using only formic acid and carbon dioxide was developed using the above FC9 strain, thereby improving the growth rate and maximum cell density of the recombinant strain. To maintain the formic acid concentration and pH of the culture medium at a constant level, 50% formic acid and 20 g / L ammonium chloride solution were automatically supplied using pH stat mode to maintain the formic acid concentration (2–3 g / L) and pH (7.2) in the medium at optimal levels for cell culture during the culture period, and the stirring speed was fixed at 400 rpm to mitigate the effect of shear stress caused by aeration. Furthermore, the initial supply amounts of air and carbon dioxide were increased from 450 cc / min and 50 cc / min, respectively, to 900 cc / min and 100 cc / min, respectively, after the logarithmic growth phase (culture time approximately 100 hours), and the antifoam agent antifoam 204 was added at a concentration of 0.01% to remove foam that appeared during the culture process.

[0113] In the above-described optimized culture method, the FC9 strain achieved growth from an initial cell density of OD600 1.32 to OD600 27.7 after 242.3 hours of culture, resulting in an approximately 2.7-fold increase in production compared to the highest cell density of OD600 10.2 under previous conditions (Fig. 8).

[0114]

[0115] The present invention relates to a method for producing a useful substance by introducing or amplifying genes coding for alanine-glyoxylate aminotransferase 1 and L-serine ammonia-lyase, which are involved in glycine supply, into a recombinant microorganism and culturing the said recombinant microorganism in a culture medium that provides only formic acid and carbon dioxide as carbon sources. According to the present invention, the assimilation efficiency of formic acid and carbon dioxide can be improved, thereby increasing the cell growth rate and maximum cell density. This can be usefully utilized to ensure the economic feasibility of the biological conversion of various high-value compounds from formic acid and carbon dioxide.

[0116]

[0117] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0118]

[0119] I have attached the electronic file.

Claims

1. To host microorganisms having a central carbon assimilation pathway, A gene encoding alanine-glyoxylate aminotransferase 1; and / or Recombinant microorganisms with enhanced carbon dioxide and formic acid assimilation, in which a gene encoding L-serine ammonia-lyase is introduced or amplified.

2. The recombinant microorganism according to claim 1, characterized in that the host microorganism i) has an internal central carbon assimilation circuit; or ii) has an externally introduced central carbon assimilation circuit.

3. In Paragraph 1, A recombinant microorganism characterized in that the gene encoding the alanine-glyoxylate aminotransferase 1 comprises the sequence of SEQ ID NO.

1.

4. In Paragraph 1, A recombinant microorganism characterized in that the gene coating the above L-serine ammonia degrading enzyme comprises the sequence of SEQ ID NO.

2.

5. In Paragraph 1, The gene encoding pyridine nucleotide transhydrogenase is overexpressed, and A recombinant microorganism characterized by the deletion of the glucose phosphotransferase system constituent protein coding gene (ptsG).

6. In Paragraph 5, A recombinant microorganism characterized in that the gene encoding the above-mentioned pyridine nucleotide transhydrogenase is the pntA or pntB gene.

7. In Paragraph 5, A recombinant microorganism characterized in that the gene encoding the above-mentioned pyridine nucleotide transhydrogenase is overexpressed by any one strong promoter selected from the group consisting of trc promoter, tac promoter, T7 promoter, lac promoter, and trp promoter.

8. In Paragraph 5, A recombinant microorganism characterized in that the gene coding for the glucose phosphotransferase system constituent protein comprises the sequence of SEQ ID NO.

9.

9. In Paragraph 1, A recombinant microorganism further comprising one or more genes encoding for one or more selected from the group consisting of formate-tetrahydrofolate ligase, methenyl tetrahydrofolate cyclohydrolase, methylene-tetrahydrofolate dehydrogenase, formate dehydrogenase, and formate dehydrogenase mutant.

10. In Paragraph 9, The above-mentioned formate-tetrahydrophorate ligase-coding gene includes the sequence of SEQ ID NO. 10; The above-mentioned methenyltetrahydrophorate cyclohydrolase coding gene includes the sequence of SEQ ID NO. 11; The above methylene-tetrahydrophorate dehydrogenase comprises the sequence of SEQ ID NO. 12; The above formic acid dehydrogenase comprises the sequence of SEQ ID NO. 13; or A recombinant microorganism characterized by the above-mentioned formic acid dehydrogenase variant comprising the sequence of SEQ ID NO.

14.

11. A recombinant microorganism according to claim 9, characterized in that the gene is cloned into and introduced into a vector comprising a replication origin having copy numbers 1 to 5.

12. The recombinant microorganism according to claim 1, characterized in that the host microorganism is selected from the group consisting of the genera Escherichia, Mannheimia, Rhodobacter, and Methylobacterium.

13. A method for manufacturing a useful substance comprising the following steps: (a) a step of producing a useful substance by culturing a recombinant microorganism according to any one of claims 1 to 12 using formic acid and carbon dioxide as carbon sources; and (b) A step of recovering the useful material generated above.

14. In Paragraph 13, A method for manufacturing characterized by step (a) of culturing recombinant microorganisms in a culture medium containing 0.2 μM to 20 μM of sodium selenite.

15. In Paragraph 13, A manufacturing method characterized by maintaining the formic acid at a concentration of 2 to 3 g / l and maintaining the pH at 6.6 to 7.0 in step (a) above, and supplying formic acid and ammonium chloride.

16. A manufacturing method according to claim 13, characterized by increasing the initial air and carbon dioxide supply amounts in step (a) from 450 cc / min and 50 cc / min, respectively, to a final 900 cc / min and 100 cc / min, respectively, while culturing.

17. In paragraph 13, the useful substance is butanol, isobutanol, 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-isobutanol, putrescine, L-ornithine, arginine, polycyclic aromatic hydrocarbon (PHA), polylactate, polylactate-co-glycolate, polyisovalerate, polyhydroxybutyrate (PHB), 4-hydroxybutyrate, biodiesel, gasoline, olefin, 5-aminovalerateric acid, gamma-aminobutyric acid, 3-hydroxyspiropionic acid, 3-aminopropionic acid, acrylic acid, A method of preparation characterized by being selected from the group consisting of 1,3-diaminopropane, caprolactam, threonine, valine, isoleucine, fumaric acid, malic acid, succinic acid, ceramide, astaxanthin, silibinin, lycopene, lutein, and retinol.

Images