Mutant microorganisms for succinic acid production into which magnesium transporters have been introduced, and a method for producing succinic acid using the same.

Abstract

The present invention relates to a mutant microorganism for succinic acid production into which a magnesium transporter has been introduced, and to a method for producing succinic acid using the same. More specifically, the present invention relates to a mutant microorganism for succinic acid production into which a gene encoding a magnesium transporter has been introduced, characterized by improving the utilization of magnesium ions by using an optimized neutralizing agent, and to a method for producing succinic acid using the same.

Description

Technical Field

[0001] The present invention relates to a mutant microorganism for producing succinic acid into which a magnesium transporter has been introduced, and a method for producing succinic acid using the same. More specifically, the present invention relates to a mutant microorganism for producing succinic acid into which a gene encoding a magnesium transporter, which is characterized by increasing the utilization of magnesium ions using an optimized neutralizing agent, has been introduced, and a method for producing succinic acid using the same.

Background Art

[0002] In recent years, in the midst of growing attention to environmental issues, efforts have been actively made to replace the production of useful compounds based on conventional fossil fuels. Along with this, research on producing bio-based succinic acid from renewable biomass has been advanced worldwide. Succinic acid is a dicarboxylic acid having 4 carbon atoms, and is used as a precursor of compounds with excellent industrial value such as 1,4-butanediol, γ-butyrolactone, diethyl succinate, N-methyl-2-pyrrolidone, and tetrahydrofuran, and is also a useful compound that may be used as a monomer of various polymers. The importance of such succinic acid has been highlighted, and many methods for producing succinic acid on a bio-based basis using various microorganisms such as Actinobacillus succinogenes, Anaerobiospirillum succiniciproducens, Escherichia coli, Mannheimia succiniciproducens, Saccharomyces cerevisiae, and Yarrowia lipolytica have been studied.

[0003] Improvement of succinic acid-producing strains has been carried out by altering the metabolic flux of the strain. The strategy was to enhance the flux to succinic acid by strengthening carbon metabolism and eliminating or reducing the production of by-products. In other words, succinic acid production is increased by introducing or strengthening genes involved in succinic acid metabolic flux, or by deleting or weakening genes that inhibit succinic acid metabolic flux. Studies have been conducted using such improved succinic acid-producing strains to adjust the optimal pH of the culture medium by performing fed-batch fermentation with many neutralizing agents (NH4OH, Ca(OH)2, Mg(OH)2, NaOH). These neutralizing agents have a considerable effect on cellular metabolism and carbon source utilization. In particular, Mg(OH)2 has been selected as a suitable neutralizing agent for succinic acid production in previously reported studies for the following reasons. Mg(OH)2 exhibits a higher carbon source utilization rate than other neutralizing agents such as Ca(OH)2, KOH, and NaOH. Due to its low solubility, magnesium citrate reduces the osmotic pressure and feedback inhibitory effect on enzymes such as succinate ions and phosphoenolpyruvate carboxykinase (PCKA), thereby promoting succinic acid production. Furthermore, Mg(OH)2 is utilized as a buffer to prevent microbial acidification. In particular, the magnesium ion itself is known to play diverse roles in cellular systems. Magnesium ions are involved in almost all metabolic pathways, playing a crucial role in structural integrity and genomic stability, and function as a cofactor for DNA repair proteins, and as an intracellular regulator of the cell cycle and apoptosis. Magnesium ions also act as essential cofactors for enzymes involved in succinic acid production, including NADPH oxidase and PCKA. Therefore, taking into account the importance of magnesium ion supply, research has been conducted to modify the magnesium transport system in E. coli to increase the absorption rate of magnesium in order to increase succinic acid production {Bioresource Technology 170(2014)125-131}.

[0004] Against this technical backdrop, the inventors of this application confirmed that it is possible to increase magnesium ion absorption by introducing many magnesium transporters and develop a high-performance succinic acid-producing bacterial strain, thus completing the present invention. [Overview of the project] [Problems that the invention aims to solve]

[0005] The object of the present invention is to provide a mutant microorganism with enhanced succinic acid production ability.

[0006] The object of the present invention is to provide a method for maximizing succinic acid production ability using the aforementioned mutant microorganism. [Means for solving the problem]

[0007] To achieve the above objective, the present invention provides a mutant microorganism in which a gene encoding a magnesium transporter has been introduced into a microorganism capable of producing succinic acid.

[0008] Furthermore, the present invention provides a method for producing succinic acid, which includes the following steps. (a) A step of culturing the mutant microorganism to produce succinic acid, and (b) A step of recovering the succinic acid generated. [Brief explanation of the drawing]

[0009] [Figure 1] These are growth and metabolite production curves for M. succiniciproducens PALK strain in a fed-batch culture using glucose as the sole carbon source, with (a) Ca(OH)2, (b) KOH, (c) NaOH, (d) Mg(OH)2, (e) NH4OH, and (f) a mixture of 1.57 M NH4OH and 6.84 M Mg(OH)2 used as neutralizing agents. [Figure 2]These are growth and metabolite production curves for M. succiniciproducens PALK strain in fed-batch culture using glucose as the sole carbon source, with the following neutralizing agents used: (a) 6.15 M NH4OH and 1.79 M Mg(OH)2, (b) 4.1 M NH4OH and 3.56 M MgOH2, (c) 3.08 M NH4OH and 5.35 M MgOH2, (d) 1.45 M NH4OH and 7.15 M Mg(OH)2, (e) 1.57 M NH4OH and 6.84 M Mg(OH)2, and (f) 0.96 M NH4OH and 7.85 M Mg(OH)2. [Figure 3] These are two-dimensional gel images showing the physiological changes of the PALK strain of M. succiniciproducens due to Mg(OH)2. (a) Two-dimensional images of the proteome of the entire cell during the logarithmic growth phase and (b) the quiescent phase when NH4OH was used as a neutralizing agent, and (c) Two-dimensional images of the proteome of the entire cell during the logarithmic growth phase and (d) the quiescent phase when a mixture of NH4OH and Mg(OH)2 was used as a neutralizing agent. [Figure 4] (a) PALKcorAKO and (b) PALK(pMS3-corA) strains, which were used to validate the magnesium transporter system in M. succiniciproducens, show growth and metabolite production curves in fed-batch culture using glucose as the sole carbon source; (c) is a schematic diagram of magnesium transporter introduction into M. succiniciproducens; and (d) is a comparative graph of intracellular magnesium ion concentrations in strains into which many magnesium transporter genes have been introduced. [Figure 5] (a) PALK(pMS3-mgtA), (b) PALK(pMS3-mgtB), (c) PALK(pMS3-mgtA-corA), (d) PALK(pMS3-corA-mgtB), and (e) PALK(pMS3-mgtA-mgtB) strains are shown as growth and metabolite production curves in fed-batch culture using glucose as the sole carbon source, and (f) is a comparative graph of succinic acid concentration and production capacity of the constructed strains. [Figure 6]These are the growth and metabolite production curves for the M. succiniciproducens PALK (pMS3-mgtB) strain in a fed-batch culture in which (a) glucose and glycerol were used together as carbon sources and (b) high concentrations of cells were inoculated. [Figure 7] These are growth and metabolite production curves for fed-batch culture of M. succiniciproducens PALK (pMS3-mgtB) strain, using glucose as the sole carbon source, with fermentation media compositions consisting of (a) 0CDM, (b) 1CDM, (c) 2CDM, (d) 0CDM and 20 g / L corn steep liquor, (e) 1CDM and 20 g / L corn steep liquor, and (f) 2CDM and 20 g / L corn steep liquor. [Figure 8] This is a graph showing the amino acid analysis results for corn steep liquor. [Figure 9] These are the growth and metabolite production curves for M. succiniciproducens PALKmgtB strain in antibiotic-free fed-batch culture using glucose as the sole carbon source. [Figure 10] This graph compares the growth and increase in succinic acid production in M. succinicinus MBEL55E (pMS3-mgtB), LPK (pMS3-mgtB), LPK7 (pMS3-mgtB), and PALFK (pMS3-mgtB) strains after 16 hours of culture using glucose as the sole carbon source. [Modes for carrying out the invention]

[0010] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by experts skilled in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0011] The inventors of the present application searched for a method to significantly improve the productivity of succinic acid compared to the prior art and examined the effect of magnesium ions in the production of succinic acid. In M. succiniciproducens, the effect of various neutralizing agents on the productivity of succinic acid was verified, and the composition of the neutralizing agent was optimized. Furthermore, an analysis of the physiological effect of magnesium ions was performed, and a gene functioning as a magnesium transporter was discovered.

[0012] Based on this, the present invention relates to a mutant microorganism into which a gene encoding a magnesium transporter has been introduced in a microorganism having the ability to produce succinic acid.

[0013] In one embodiment, the gene encoding the magnesium transporter may be one or more or two or more selected from the group consisting of CorA, MgtA, and MgtB, but is not limited thereto.

[0014] Specifically, the gene encoding the magnesium transporter may include, but is not limited to, the following. (1) CorA, MgtA or MgtB gene, (2) CorA and MgtA genes, (3) CorA and MgtB genes, or (4) MgtA and MgtB genes.

[0015] In one embodiment, the CorA gene may include the sequence of SEQ ID NO: 17.

[0016] [SEQ ID NO: 17] JPEG2026525362000002.jpg70170

[0017] In other embodiments, the MgtA gene may include the sequence of SEQ ID NO: 18.

[0018] [SEQ ID NO: 18] JPEG2026525362000003.jpg215170

[0019] In yet another embodiment, the MgtB gene may include the sequence of SEQ ID NO: 19.

[0020] [Sequence ID 19] JPEG2026525362000004.jpg214170

[0021] The aforementioned mutant microorganisms may also have further deletions of antibiotic resistance genes. Specifically, they may be characterized by further deletions of the antibiotic resistance gene of SEQ ID NO: 40 or SEQ ID NO: 41. This makes it possible to culture them in a culture medium without the addition of antibiotics.

[0022] [Sequence ID 40] JPEG2026525362000005.jpg106170

[0023] [Sequence ID 41] JPEG2026525362000006.jpg128170

[0024] In this invention, "overexpression" refers to an expression level higher than the level at which the gene in question is normally expressed in a cell. This concept includes methods such as replacing the promoter of a gene present in the genome with a strong promoter, or increasing the expression level by cloning the gene in question into an expression vector and transforming the cell.

[0025] In the present invention, “vector” means a DNA product containing a DNA sequence operably linked to a suitable regulatory sequence that can express DNA in 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 can replicate and function independently of the host genome, or, in some cases, be incorporated into the genome itself. Since plasmids are currently the most commonly used form of vector, the terms “plasmid” and “vector” in this specification are used interchangeably as they may be. For the purposes of the present invention, plasmid vectors are preferred. A typical plasmid vector that can be used for such purposes has a structure that includes (a) a replication origin that enables efficient replication to include several hundred plasmid vectors per host cell, (b) an antibiotic resistance gene that enables selection of host cells transformed by the plasmid vector, and (c) restriction enzyme cleavage sites into which foreign DNA fragments can be inserted. Even when suitable restriction enzyme cleavage sites are not available, the vector and foreign DNA can be easily ligated using a conventionally synthesized oligonucleotide adapter or linker.

[0026] After ligation, the vector is transformed into a suitable host cell. The succinic acid-producing microorganism may be characterized by being selected from the group consisting of the genera Mannheimia sp., Actinobacillus sp., Anaerobiospirillum sp., Basfia sp., Escherichia coli, and Corynebacterium sp. Furthermore, the genus Mannheimia includes the genus Basfia, which has recently been found to be substantially the same genus.

[0027] Preferably, the microorganism having succinic acid production ability may be characterized by being Mannheimia succiniciproducens PALK (KCTC10973BP).

[0028] In some cases, this may include Basfia mutant strains that are very similar to Mannheimia succiniciproducens but have been given genes encoding magnesium transporters.

[0029] Nucleic acids are "operably linked" when they are arranged in a functional relationship with other nucleic acid sequences. This means that genes and regulatory sequences are linked in such a way that gene expression becomes possible when the appropriate molecule (e.g., a transcription-activating protein) binds to the regulatory sequence. For example, DNA for a pre-sequence or secretion leader is operably linked to the DNA for the polypeptide if it is expressed as a precursor protein involved in polypeptide secretion; promoters or enhancers are operably linked to coding sequences if they affect the transcription of the sequence; ribosome binding sites are operably linked to coding sequences if they affect the transcription of the sequence; or ribosome binding sites are operably linked to coding sequences if they are positioned to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are in contact and, in the case of secretion leaders, are in contact with each other within the reading frame. However, enhancers do not need to be in contact. Linking of these sequences is achieved by ligation at appropriate restriction enzyme sites. If such a site does not exist, a synthetic oligonucleotide adapter or linker using conventional methods is used.

[0030] As used herein, the term "expression vector" typically refers to a recombinant carrier into which a heterologous DNA fragment is inserted, and generally means a double-stranded DNA fragment. Here, heterologous DNA refers to heterologous DNA that is not naturally found in the host cell. If present in the host cell, the expression vector can replicate independently of the host chromosomal DNA, and multiple copies of the vector and the (heterologous) DNA inserted into them may be produced.

[0031] As is well known in this field, in order to increase the expression level of a transdextrin in a host cell, the gene must be operably ligated to a transcriptional and translational regulatory sequence that functions within a selected expression host. Preferably, the regulatory sequence and the gene are contained within a single expression vector that also contains a bacterial selection marker and an origin of replication. If the host cell is a eukaryotic cell, the expression vector preferably further contains an expression marker useful in the eukaryotic expression host.

[0032] Host cells transformed or transduced by the expression vector constitute another aspect of the present invention. As used herein, the term “transformation” means introducing DNA into a host so that the DNA becomes replicable as an extrachromosomal factor or by integration into a chromosome. As used herein, the term “transduction” means that an expression vector is received by a host cell, regardless of whether any coding sequence is actually expressed.

[0033] Of course, it should be understood that not all vectors and regulatory sequences function equally well to express the DNA sequence of the present invention. Similarly, not all hosts function equally well with the same expression system. However, those skilled in the art can appropriately select from multiple vectors, regulatory sequences, and hosts without excluding the scope of the present invention and without requiring an excessive experimental burden. For example, when selecting a vector, the host must be considered because the vector must be replicated within it. The copy number of the vector, its ability to regulate copy number, and the expression of other proteins encoded by the vector, such as antibiotic markers, must also be considered. When selecting a regulatory sequence, various factors must also be considered. For example, the relative strength of the sequence, its regulatorability, and its compatibility with the DNA sequence of the present invention must be considered, particularly in relation to potential secondary structures. Single-cell hosts must be selected considering factors such as the toxicity of the product encoded by the selected vector and the DNA sequence of the present invention, its secretory characteristics, its ability to accurately fold the protein, its culture and fermentation requirements, and the ease with which the product encoded by the DNA sequence of the present invention can be purified from the host. Within the range of these variables, those skilled in the art can select various vector / expression regulatory sequence / host combinations that can be expressed by fermenting the DNA sequence of the present invention or in large-scale animal culture.

[0034] Based on this, the present invention relates in other aspects to a method for producing succinic acid, comprising the steps of (a) culturing the mutant microorganism to produce succinic acid, and (b) recovering the produced succinic acid.

[0035] The culture may be characterized by using glucose and / or glycerol as a carbon source.

[0036] The culture may, but is not limited to, be carried out under anaerobic conditions.

[0037] The culture may be characterized by being carried out in a medium containing corn steep liquor. In some cases, a chemically defined medium (CDM) may also be included.

[0038] The culture may be carried out by adding a neutralizing agent selected from the group consisting of Ca(OH)2, KOH, NaOH, Mg(OH)2, NH4OH, and two or more of these. The neutralizing agent was selected as one that has a significant effect on the cell's metabolism and carbon source utilization and is suitable for succinic acid production.

[0039] When using the aforementioned neutralizing agent, it may be included as Ca(OH)2, KOH, NaOH, Mg(OH)2, NH4OH, or a mixture of NH4OH and Mg(OH)2.

[0040] The aforementioned succinic acid-producing microorganisms may be characterized by being selected from the group consisting of the genera Mannheimia sp., Actinobacillus sp., Anaerobiospirillum sp., Basfia sp., Escherichia coli, and Corynebacterium sp. Furthermore, the genus Mannheimia includes the genus Basfia, which has recently been identified as substantially the same genus.

[0041] The microorganism having succinic acid production ability may be, for example, Mannheimia succiniciproducens.

[0042] The microorganism having succinic acid production ability may be Mannheimia succiniciproducens MBEL55E (KCTC0769BP).

[0043] The inventors identified the M. succiniproducens MBEL55E (KCTC0769BP) strain, which produces succinic acid with high efficiency from Korean beef, and have published their findings on the genome sequence and metabolic characteristics of the strain (Hong et al., Nature Biotechnol., 22:1275, 2004). Furthermore, the inventors created a mutant strain, M. succiniproducens LPK (KCTC10558BP), in which the lactate dehydrogenase gene (ldhA) and pyruvate-formate dehydrogenase gene (pfl) were deleted in order to suppress the production of lactic acid and formic acid in M. succiniproducens MBEL55E, a type of rumen bacterium.

[0044] The aforementioned succinic acid-producing microorganism may be characterized by being LPK (KCTC10558BP), which is Mannheimia succiniciproducens MBEL55E in which the gene encoding lactate dehydrogenase (ldhA) and the gene encoding pyruvate-formate hydrolase (pfl) are deleted.

[0045] The aforementioned succinic acid-producing microorganism may be characterized by being Mannheimia succiniciproducens PALK (KCTC10973BP), a mutant rumen bacterium produced by deleting the gene encoding lactate dehydrogenase (ldhA), the gene encoding phosphotransacetylase (pta), and the gene encoding acetate kinase (ackA) in Mannheimia succiniciproducens, without deleting the gene encoding pyruvate-formate dehydrogenase (pfl).

[0046] The inventors of this invention have identified a strain of M. succiniciproducens MBEL55E (KCTC0769BP) that produces succinic acid with high efficiency from Korean beef, and have published the genome sequence and metabolic characteristics of the strain (Hong et al., Nature Biotechnol., 22:1275, 2004). Furthermore, the inventors created a mutant strain, M. succiniproducens LPK (KCTC10558BP), in which the lactate dehydrogenase gene (ldhA) and pyruvate-formate dehydrogenase gene (pfl) were deleted in M. succiniproducens MBEL55E, a type of rumen bacterium, in order to suppress the production of lactic acid and formic acid. In addition, to inhibit the production of acetic acid, they created a mutant strain, M. succiniproducens LPK7 (KCTC10626BP), in which the phosphotransacetylase gene (pta) and acetate kinase gene (ackA) were deleted in the aforementioned mutant strain M. succiniproducens LPK. Succinic acid was produced by culturing these mutant strains under anaerobic conditions (International Publication No. WO05 / 052135).

[0047] Based on the genomic information of the Mannheimia succiniciproducens 55E strain, a type of rumen bacterium, the lactate dehydrogenase gene (ldhA) and the pyruvate-formate dehydrogenase gene (pfl) were identified. Then, using a gene deletion vector, these two genes were deleted from the Mannheimia succiniciproducens 55E genome to create a mutant strain [Mannheimia sp. LPK (KCTC 10558BP)] (Korean Patent No. 10-0556099).

[0048] The succinic acid-producing microorganism may be Mannheimia succiniciproducens LPK7 (KCTC10626BP), which is a mutant strain of Mannheimia succiniciproducens LPK in which the phosphotransacetylase gene (pta) and the acetate kinase gene (ackA) are deleted (Korean Patent No. 10-0630819).

[0049] The succinic acid-producing microorganism may be Mannheimia succiniciproducens PALK (KCTC10973BP), a mutant microorganism that, under anaerobic conditions, produces almost no other organic acids besides succinic acid and produces only succinic acid at high concentrations, while not deleting the gene encoding pyruvate-formate hydrolysate (pfl) in Mannheimia succiniciproducens, and is produced by deleting the gene encoding lactate dehydrogenase (ldhA), the gene encoding phosphotransacetylase (pta), and the gene encoding acetate kinase (ackA) (Korean Patent No. 10-0780324).

[0050] The microorganism having succinic acid production ability may be M. succinicinproducens PALFK (KCTC11694BP), a strain of Mannheimia succinicinproducens PALK (KCTC10973BP) that has the ability to metabolize sucrose and glycerol simultaneously by deleting the gene encoding fructose phosphotransferase (fruA) in the genomic DNA (International Publication No. WO2012030130).

[0051] In this invention, the fermentation process was optimized to produce an excess of succinic acid. In particular, selecting the most appropriate neutralizing agent to maintain the optimal pH (pH 6.5) of M. succiniproducens is important for increasing succinic acid production. Therefore, in order to search for the most effective neutralizing agent for succinic acid production, a total of five agents were selected: NH4OH, KOH, Ca(OH)2, Mg(OH)2, and NaOH, and M. succiniproducens PALK (KCTC10973BP) was selected as the platform strain. The M. succiniproducens PALK (KCTC10973BP) strain is a strain created by deleting the genes encoding lactate dehydrogenase, phosphotransacetylase, and acetate kinase in the wild-type M. succiniproducens strain. It is a succinic acid-producing mutant microorganism that produces only succinic acid at high concentrations, while producing almost no other organic acids under anaerobic conditions. First, in fed-batch fermentation using 8.4 M Ca(OH)2 as a neutralizing agent, 16.79 g / L of succinic acid was obtained with a yield of 0.67 g / L / h, with a productivity of 0.56 mol / mol relative to glucose (Figure 1; Table 1). However, the formation of a calcium precipitate was observed immediately after the addition of Ca(OH)2, because calcium ions are rapidly converted into calcium carbonate, a water-insoluble white precipitate, along with carbonic acid in the culture medium. Such precipitate formation is undesirable because carbonic acid must be continuously supplied in M. succinic acid (1 mole of carbonic acid is consumed to produce 1 mole of succinic acid). Therefore, considering the low productivity of succinic acid and the formation of calcium carbonate precipitate, it was confirmed that Ca(OH)2 is not a suitable neutralizing agent. Next, KOH and NaOH, which have strong basic properties, were used as neutralizing agents.In fed-batch fermentation using KOH as a neutralizing agent, 25.53 g / L of succinic acid was obtained with a yield of 0.93 mol / mol and a productivity of 0.88 g / L / h relative to glucose, respectively. In contrast, with NaOH, 33.16 g / L of succinic acid was obtained with a yield of 1.02 mol / mol and a productivity of 1.15 g / L / h relative to glucose (Figure 1; Table 1). However, cell aggregation problems were observed when KOH or NaOH were used. This is because the presence of high concentrations of potassium and sodium ions in the fermentation medium creates a high osmotic pressure environment, which can damage the microbial morphology. As a result, it became clear that using KOH or NaOH as a neutralizing agent resulted in low cell growth and low succinic acid production capacity. Furthermore, NH4OH is an intermediate basic solution widely used in industry as a nitrogen source for microorganisms and is used for pH adjustment during fermentation. In fed-batch fermentation using 28% w / w NH4OH as a neutralizing agent, 58.6 g / L of succinic acid was obtained with a yield of 1.04 mol / mol and a productivity of 2.44 g / L / h relative to glucose (Figure 1; Table 1).

[0052] To further increase succinic acid production, Mg(OH)2 has been considered as a neutralizing agent for several reasons. First, it has been reported that using Mg(OH)2 instead of KOH, Ca(OH)2, and NaOH improves carbon utilization. Also, because magnesium succinate has lower solubility compared to other succinates such as sodium succinate and potassium succinate, feedback inhibition by succinate anions is reduced when Mg(OH)2 is used. Furthermore, it has been reported that Mg(OH)2 exhibits high buffering capacity and inherent insolubility under excess supply conditions. Therefore, even when Mg(OH)2 is supplied in excess, it does not cause a rapid increase in pH that could impair the fermentation process. Rather, the insoluble Mg(OH)2, in combination with the nutrients and biosolid matrix present in the fermenter, provides a gradual increase in pH and high buffering capacity. Furthermore, the magnesium ion in Mg(OH)2 itself is known to play diverse roles in cellular systems, including structural integrity, genomic stability, DNA repair, regulation of the cell cycle and apoptosis, and, most importantly, succinic acid production. However, in fed-batch fermentation using only Mg(OH)2 as a neutralizing agent, the formation of magnesium carbonate precipitate was observed, yielding 33.72 g / L of succinic acid with a yield of 1.04 mol / mol relative to glucose and a productivity of 1.37 g / L / h (Figure 1; Table 1). Therefore, mixtures of NH4OH and Mg(OH)2 were tested at various concentrations (6.15M NH4OH and 1.79M Mg(OH)2, 4.1M NH4OH and 3.56M MgOH2, 3.08M NH4OH and 5.35M MgOH2, 1.45M NH4OH and 7.15M Mg(OH)2, 1.57M NH4OH and 6.84M Mg(OH)2, and 0.96M NH4OH and 7.85M Mg(OH)2) to determine the optimal amount of each component (Figure 2; Table 1). It was confirmed that both cell growth and succinic acid production improved as the fraction of Mg(OH)2 in the mixture increased. This improvement began to saturate when the concentration of Mg(OH)2 in the mixture exceeded 6.84M, and a slight decrease in succinic acid productivity was observed.Furthermore, compared to fermentation using NH4OH alone, a significant increase in cell concentration (35% increase) and succinic acid production (27% increase) was observed. Therefore, an optimized mixture (1.57 M NH4OH and 6.84 M Mg(OH)2) was used as an optimized neutralizing agent for M. succiniciproducens in this invention.

[0053] To investigate the factors contributing to improved cell growth and increased succinic acid production through the use of Mg(OH)2, the physiological changes of the M. succiniproducens PALK (KCTC10973BP) strain were analyzed. Whole cell proteome samples were collected during the logarithmic growth phase (10 hours after the start of fermentation) and the quiescent phase (16 hours after the start of fermentation) of the M. succiniproducens PALK (KCTC10973BP) strain during fed-batch fermentation (Figure 3). Based on a proteome reference map and more than 200 proteins identified and characterized in previously published studies (Lee et al., Bioprocess Biosyst Eng 2010, 33:97-107), the intensity of protein spots detected on a two-dimensional gel was evaluated (Figure 3). Based on the intensity and area comparison of the analyzed spots, the difference in the number of spots obtained during the logarithmic growth phase and the quiescent phase was calculated (Table 2).

[0054] As expected, enzymes involved in succinic acid production, such as PckA and FumC, increased in both the logarithmic growth phase and the quiescent phase when a mixture of NH4OH and Mg(OH)2 was used as a neutralizing agent, compared to fed-batch fermentation using only NH4OH. Furthermore, we found increases in the enzymes mentioned above (PckA, fructose kinase, enolase, glyceraldehyde 3-phosphate dehydrogenase, 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase), TCA cycle enzymes (fumarate hydratase, malate dehydrogenase), sugar-binding proteins (phosphomannomutase), and phosphate transferase systems (phosphocarrier protein HPr, sugar-specific IIA component). In particular, the ploidy change was more pronounced during the logarithmic growth phase, confirming the importance of Mg(OH)2 for stable cell growth. Additionally, due to active metabolism during the logarithmic growth phase, a higher cell density was observed when a mixture of NH4OH and 6.84M Mg(OH)2 was used as a neutralizing agent.

[0055] As confirmed in the aforementioned study, the use of Mg(OH)2 as a neutralizing agent has been shown to be beneficial for succinic acid production. Therefore, the intracellular magnesium ion concentration of the M. succiniproducens PALK (KCTC10973BP) strain was analyzed (Figure 4; Tables 3-4). The intracellular magnesium ion concentration in the PALK strain (3.452 mM) was found to be significantly higher than in other strains such as Escherichia coli and Bacillus subtilis (1-2 mM). Furthermore, the genome sequence of M. succiniproducens was investigated to examine the magnesium transport system in M. succiniproducens. According to a previously published study (Hong et al., Nat. Biotechnol., 2004, 22:1275-1281), genome analysis of the wild-type M. succiniciproducens MBEL55E (KCTC0769BP) strain revealed that this selective anaerobic bacterium possesses the corA gene as a magnesium transporter, while the mgtA and mgtB genes, known as independent magnesium transporters like corA, are absent from its genome. Furthermore, to investigate the importance of the corA gene, a PALKcorAKO strain was constructed by deleting the corA gene from the genome of the PALK strain. As a result of fed-batch fermentation, it was confirmed that the cells hardly grew (Figure 4). Therefore, to clarify the effect of increased intracellular magnesium ion concentration on cell growth and succinic acid production, the corA gene was overexpressed in the PALK strain. As a result, we found that the PALK(pMS3-corA) strain took up more magnesium ions and had a higher intracellular magnesium ion concentration (5.814 mM) compared to the PALK strain (3.452 mM). In fed-batch fermentation, 86.66 g / L of succinic acid was obtained with a yield of 1.23 mol / mol relative to glucose and a productivity of 3.30 g / L / h (Figure 4; Table 5).

[0056] In fact, to improve succinic acid production by introducing magnesium transporters, several genes encoding magnesium transporters in other microorganisms were introduced into the M. succinicin-producens PALK (KCTC10973BP) strain. First, the mgtA gene, which acts as a magnesium transporter in E. coli, was introduced. The PALK(pMS3-mgtA) strain thus developed took up a large amount of magnesium ions, and its intracellular magnesium ion concentration (6.240 mM) was higher than that of the PALK strain (3.452 mM), but similar to that of the PALK(pMS3-corA) strain (5.814 mM) (Figure 4; Tables 3-4). In fed-batch fermentation of the PALK(pMS3-mgtA) strain, 82.33 g / L of succinic acid was obtained with a yield of 1.19 mol / mol relative to glucose and a productivity of 3.13 g / L / h (Figure 5; Table 5). Next, the mgtB gene, which acts as a magnesium transporter in Salmonella enterica, was also introduced into the PALK strain. The PALK(pMS3-mgtB) strain developed in this way took up a large amount of magnesium ions and had the highest intracellular magnesium ion concentration (8.474 mM) among the M. succinicinus strains (Figure 4; Tables 3-4). Furthermore, in fed-batch fermentation of the PALK(pMS3-mgtB) strain, 94.25 g / L of succinic acid was obtained with a yield of 1.26 mol / mol relative to glucose and a productivity of 3.46 g / L / h (Figure 5; Table 5). In addition, to confirm the synergistic effect of simultaneously overexpressing multiple magnesium transporters, three strains were constructed in which two different magnesium transporters were overexpressed. In the fed-batch fermentation of the PALK(pMS3-mgtA-corA), PALK(pMS3-corA-mgtB), and PALK(pMS3-mgtA-mgtB) strains developed in this manner, succinic acid was obtained in concentrations of 83.13 g / L, 94.12 g / L, and 76.69 g / L, respectively, with yields of 1.26 mol / mol, 1.26 mol / mol, and 1.19 mol / mol relative to glucose, and productivitys of 3.29 g / L / h, 3.45 g / L / h, and 3.03 g / L / h, respectively (Figure 5; Table 5).As a result, it was confirmed that succinic acid production did not increase significantly in M. succinicin strains that overexpressed different magnesium transporters.

[0057] Based on the studies on the introduction of heterologous magnesium transporters, the PALK(pMS3-mgtB) strain exhibits higher succinic acid titer, yield, and productivity compared to other strains. Therefore, the culture conditions were optimized using the PALK(pMS3-mgtB) strain. To further improve succinic acid production by the constructed PALK(pMS3-mgtB) strain, fed-batch fermentation was performed in restriction medium using both glucose and glycerol as carbon sources. Glycerol is known to be advantageous for succinic acid production because it provides twice the reducing equivalent per mole compared to glucose (Ahn et al., Nat.Commun., 2020, 11:1970). When the PALK(pMS3-mgtB) strain was subjected to fed-batch fermentation using both glucose and glycerol as carbon sources in restriction medium, 110.92 g / L of succinic acid was obtained with a yield of 1.42 mol / mol of glucose (to facilitate comparison of yields, when both glucose and glycerol were used as carbon sources, the number of carbon atoms from both sources was included in the calculation and expressed in glucose equivalent. Hereafter, this notation will be consistently used as "mol / mol of glucose") and a productivity of 4.07 g / L / h (Figure 6; Table 5). This result shows an improvement compared to the results of conventional fed-batch fermentation using glucose as the sole carbon source. In addition, one of the fermentation characteristics of M. succiniciproducens is the low cell density during fed-batch fermentation. In this study, the PALK(pMS3-corA), PALK(pMS3-mgtA), PALK(pMS3-mgtB), PALK(pMS3-mgtA-corA), PALK(pMS3-corA-mgtB), and PALK(pMS3-mgtA-mgtB) strains constructed achieved maximum cell densities of 3.52 gDCW / L, 4.19 gDCW / L, 3.45 gDCW / L, 3.08 gDCW / L, 3.01 gDCW / L, and 3.58 gDCW / L, respectively, during fermentation with glucose. On the other hand, the productivity per cell for these strains was 0.94 g / gDCWh, 0.75 g / gDCWh, 1.00 g / gDCWh, 1.07 g / gDCWh, 1.15 g / gDCWh, and 0.85 g / gDCWh, respectively.Thus, because the constructed strain has high productivity per cell, its productivity is relatively high compared to other succinic acid-producing strains, even with a lower cell density. Since the cells themselves function as cellular factories, productivity can be further increased by increasing the cell density. Therefore, succinic acid was produced by inoculating the constructed PALK(pMS3-mgtB) strain at a high concentration and utilizing the improved productivity under anaerobic conditions. In a restriction medium, when cells were inoculated at an initial high concentration of 9.25 g DCW / L and fed-batch fermentation was performed using both glucose and glycerol as carbon sources, 153.23 g / L of succinic acid was obtained with a glucose yield of 1.25 mol / mol relative to glucose and a productivity of 11.7 g / L / h.

[0058] In addition, to reduce production costs, the fermentation culture conditions of the constructed strain were optimized. To lower production costs, the supplements were removed from the current fermentation medium, which contains seven amino acids and six vitamins, as developed in a previously reported study (Song et al., Appl. Microb. Biotechnol., 2008, 79:263-272). To verify the effect of supplements on cell growth and succinic acid production of M. succinicin, the PALK(pMS3-mgtB) strain was cultured in a restriction medium that did not contain amino acids and vitamins. As a result, the PALK(pMS3-mgtB) strain was unable to achieve cell growth and succinic acid production without the supplements (Figure 7). Therefore, other inexpensive supplements were considered as an alternative to the supply of purified amino acids and vitamins. Among several supplements, corn steep liquor (CSL) was selected as the alternative supplement. CSL is a byproduct of the maize wet milling industry and contains substantial amounts of amino acids, polypeptides, and B complex vitamins, making it an excellent nitrogen source for many microorganisms. Before supplying CSL to the fermentation medium, the amino acid composition of CSL was analyzed and confirmed that it contained the necessary amino acids (Figure 8). Furthermore, based on previously reported research (Yu et al., Biochem.Eng.J., 2008, 39:496-502), it was confirmed that all the necessary vitamins were contained in CSL. In fed-batch fermentation of the PALK (pMS3-mgtB) strain supplied with CSL, 41 g / L of succinic acid was obtained with a yield of 1.25 mol / mol relative to glucose and a productivity of 1.61 g / L / h (Figure 7; Table 6). In addition, cell growth was fully restored, similar to the fed-batch fermentation with 7 amino acids and 6 vitamins supplied (Figure 7; Table 6). Furthermore, we supplemented the fermentation medium with cysteine ​​and nicotinic acid, which are representative amino acids and vitamins found in restriction media, to verify whether CSL supplementation was sufficient to replace amino acids and vitamins. As a result, no significant increase in cell growth or succinic acid production was observed in fed-batch fermentation using media supplied with CSL, cysteine, and nicotinic acid, indicating that CSL can replace expensive amino acids and vitamins (Figure 7; Table 6).As a result, cell growth was fully restored by using CSL, but the final succinic acid production concentration was 43.5% of that of cultures supplied with purified amino acids and vitamins.

[0059] Furthermore, in industrial applications, the stability of metabolically engineered bacterial strains is extremely important. In particular, strain stability is crucial when using plasmid-based expression systems. Therefore, the development of antibiotic-free strains for succinic acid production is important (Choi et al., Metab.Eng., 2018, 47:463-474). To address plasmid instability, a marker gene deletion strategy was used to insert the mgtB gene into the chromosome of M. succinicin, eliminating antibiotic dependence for plasmid stability by deleting the antibiotic marker gene (Kim et al., FEMS Microbiol.Lett., 2008, 278;78-85). As a result, 94.23 g / L of succinic acid was obtained by fed-batch fermentation of the final PALKmgtB strain without antibiotic addition, with a yield of 1.26 mol / mol relative to glucose and a productivity of 3.45 g / L / h (Figure 9). Therefore, the succinic acid production index of the antibiotic-free PALKmgtB strain is similar to that of the plasmid-based strain PALK(pMS3-mgtB) (Table 5), suggesting the potential for industrial-scale succinic acid production. Furthermore, the mgtB gene, a magnesium transporter, was introduced into already established succinic acid-producing microorganisms: M. succiniciproducens MBEL55E (KCTC0769BP), LPK (KCTC10558BP), LPK7 (KCTC10626BP), and PALFK (KCTC11694BP). Tube cultures of the MBEL55E(pMS3-mgtB), LPK(pMS3-mgtB), LPK7(pMS3-mgtB), and PALFK(pMS3-mgtB) strains developed in this manner yielded succinic acid concentrations of 6.00 g / L, 7.32 g / L, 6.64 g / L, and 6.70 g / L, respectively, after 16 hours (Figure 10). [Examples]

[0060] The present invention will be described in more detail below with reference to examples. These examples are provided solely to illustrate the present invention more concretely, and it will be obvious to those ordinary skill in the art that the scope of the present invention is not limited by these examples.

[0061] In particular, in the following examples, only Mannheimia microorganisms, which are succinic acid-producing microorganisms, were used as host cells to replace or overexpress the gene according to the present invention. However, it will be obvious to those skilled in the art that mutant microorganisms having the same succinic acid-producing ability as the present invention can be obtained even when other types of succinic acid-producing microorganisms are used.

[0062] [Example 1] Preparation of CorA, MgtA, and MgtB overexpression vectors (pMS3-corA, pMS3-mgtA, pMS3-mgtB, pMS3-corA-mgtB, pMS3-mgtA-mgtB, pMS3-mgtA-corA), and preparation of PALK(pMS3-corA), PALK(pMS3-mgtA), PALK(pMS3-mgtB), PALK(pMS3-corA-mgtB), PALK(pMS3-mgtA-mgtB), PALK(pMS3-mgtA-corA), MBEL55E(pMS3-mgtB), LPK(pMS3-mgtB), LPK7(pMS3-mgtB), and PALFK(pMS3-mgtB) strains.

[0063] To overexpress the genes encoding magnesium transporters in multiple microorganisms, overexpression vectors were constructed for each. Using primers SEQ ID NOs. 1 and 2, 3 and 4, and 5 and 6, PCR was performed using genomic DNA from M. succiniciproducens, E. coli, and S. enterica as templates. The resulting PCRs were digested with EcoRI and KpnI restriction enzymes, and then cloned into the EcoRI and KpnI sites of pMS3 (Jang et al., Appl. Environ. Microb. 73(17):5411-5420., 2007) to produce the overexpression vectors pMS3-corA and pMS3-mgtA. In the case of the mgtB gene, after digestion with KpnI and PstI restriction enzymes, cloning into the KpnI and PstI sites of pMS3 was performed to produce the overexpression vector pMS3-mgtB. To simultaneously overexpress two types of magnesium transporters, separate overexpression vectors were constructed for each. PCR was performed using the pMS3-mgtB vector as a template with primers SEQ ID NOs.7 and 8. After digesting the resulting PCR with HindIII restriction enzyme, the overexpression vectors pMS3-corA-mgtB and pMS3-mgtA-mgtB were constructed by cloning the resulting PCR into the HindIII region of pMS3-corA and pMS3-mgtA, respectively. In addition, PCR was performed using the pMS3-corA vector as a template with primers SEQ ID NOs.9 and 10. After digesting the resulting PCR with HindIII restriction enzyme, the overexpression vector pMS3-mgtA-corA was constructed by cloning the resulting PCR into the HindIII region of pMS3-mgtA, respectively.

[0064] Sequence ID 1: 5'-TACAACTCTACTGGGGAGGATGATAAATGCATTTGCAC

[0065] Sequence ID 2: 5'-TCTAGAGGATCCCCGGGTACTTACAACCAGCCTTTACG

[0066] Sequence ID 3:5'-ACTTTTATCAACTCTACTGGGGAGGATGTTTAAAGAAATTTTTAC

[0067] Sequence ID 4:5'-TCTAGAGGATCCCCGGGTACTTATTGCCAGCCGTAACGAC

[0068] Sequence ID 5: 5'-GAGGAATTCGAGCTCGGTACATGACTGACATGAACATTG

[0069] Sequence ID 6: 5'-GCCAAGCTTGCATGCCTGCATTAAAACCACTGGCCAAAG

[0070] Sequence ID 7: 5'-AGTCGACCTGCAGGCATGCACTATTCTGTTGGCTAATGC

[0071] Sequence ID 8: 5'-GTCGCCCGCCAAAACAGCCATTAAAACCACTGGCCAAAG

[0072] Sequence ID 9: 5'-AGTCGACCTGCAGGCATGCACTATTCTGTTGGCTAATGC

[0073] Sequence ID 10: 5'-GTCGCCCGCCAAAACAGCCAAGCTTTTACAACCAGCCTTTACG

[0074] By introducing the pMS3-corA, pMS3-mgtA, pMS3-mgtB, pMS3-corA-mgtB, pMS3-mgtA-mgtB, and pMS3-mgtA-corA strains prepared as described above into M. succiniciproducens PALK (KCTC10973BP), the following strains were ultimately produced: PALK(pMS3-corA), PALK(pMS3-mgtA), PALK(pMS3-mgtB), PALK(pMS3-corA-mgtB), PALK(pMS3-mgtA-mgtB), and PALK(pMS3-mgtA-corA). Furthermore, by introducing pMS3-mgtB into M. succiniciproducens MBEL55E (KCTC0769BP), LPK (KCTC10558BP), LPK7 (KCTC10626BP), and PALFK (KCTC11694BP), the MBEL55E (pMS3-mgtB), LPK (pMS3-mgtB), LPK7 (pMS3-mgtB), and PALFK (pMS3-mgtB) strains were ultimately created. To introduce the overexpression vector as described above, M. succiniciproducens PALK (KCTC10973BP), MBEL55E (KCTC0769BP), LPK (KCTC10558BP), LPK7 (KCTC10626BP), and PALFK (KCTC11694BP) were spread on BHI (Brain-Heart Infusion) solid medium and cultured at 39°C for 36 hours. After that, the colonies were inoculated into 10 mL of BHI liquid medium and cultured for 12 hours. The sufficiently grown cell culture medium was again inoculated into 100 mL of BHI liquid medium at 1 mL and cultured in a static incubator at 39°C. After about 4 to 5 hours, cell growth was overexpressed. 600When the bacterial culture medium reached approximately 0.3-0.5 based on the baseline, the bacterial culture medium was left at 0°C-4°C for 20 minutes to stop cell growth, and then the cells were obtained by centrifugation at 4°C and 4,500 rpm for 15 minutes. Next, the cells were resuspended in 200 mL of 10% glycerol solution at 4°C, and then centrifuged again under the same conditions. This resuspending and centrifugation was repeated a total of three times, with the 10% glycerol solution being reduced by half each time. The finally obtained cells were resuspended in the same volume of 10% glycerol solution, dispensed, and stored at -80°C. The cell concentration suspension obtained above was mixed with the four overexpression vectors prepared in this example, and electroporation was performed under conditions of 2.5kV, 25μF, and 200Ω to attempt transformation of the vectors with M. succiniciproducens PALK (KCTC10973BP), MBEL55E (KCTC0769BP), LPK (KCTC10558BP), LPK7 (KCTC10626BP), and PALFK (KCTC11694BP). The electroporated cells were added to BHI liquid medium and recovered in a static incubator at 39°C for 1 hour. The entire culture medium was then spread onto BHI solid medium containing the antibiotic ampicillin 2.5μg / mL and cultured in a static incubator at 39°C for 48 hours or more. The mutant strains formed in the aforementioned medium were cultured in antibiotic-containing BHI liquid medium, and the vectors recovered by the vector mini-prep method were examined by electrophoresis to confirm the introduction of the overexpression vector.

[0075] [Example 2] Preparation of CorA deletion vector (pcorAKO) and preparation of PALKcorAKO strain

[0076] To evaluate the effect of the corA gene, which encodes a naturally occurring magnesium transporter in M. succiniciproducens strains, on improving succinic acid production, a pcorAKO vector was constructed by deleting the CorA-encoding gene present in the genome of the M. succiniciproducens PALK (KCTC10973BP) strain. To construct the pcorAKO, pSacHR06, containing the sacB gene, was digested with XhoI and SacI. Subsequently, the upstream and downstream sequences of the CorA-encoding gene present in the genome were analyzed using the genome of the M. succiniciproducens PALK (KCTC10973BP) strain as a template, and PCR was performed using primers SEQ ID NOs. 11 and 12, and SEQ ID NOs. 13 and 14, respectively. The lox66-cat-lox77 cassette was obtained by performing PCR using a vector containing the chloramphenicol resistance gene as a template, and primers SEQ ID NOs. 15 and 16. The resulting linear pSacHR06, the upstream and downstream sequences of the CorA-encoding gene (1 kb each), and the lox66-cat-lox77 cassette were ligated using the Gibson assembly (Gibson et al., Nat. Methods, 6(5):343, 2009) to obtain pcorAKO. The pcorAKO prepared as described above was introduced into M. succiniciproducens PALK (KCTC10973BP) to finally create the PALKcorAKO strain.

[0077] Sequence ID 11: 5'-TTCAACGGGAAACGTCTTGCATGGCTGCAGTTCTTCGG

[0078] Sequence ID 12:5'-GTGTCACCTAATTTTGCTTTTAAGCTAAAAGTGCG

[0079] Sequence ID 13: 5'-AAAGCAAAATAGGTGACACTATAGAACGC

[0080] Sequence ID 14:5'-AGTATGACAACCGCATAGGCCACTAGTG

[0081] Sequence ID 15:5'-GCCTATGCGGTTGTCATACTCCCTAGGTTC

[0082] Sequence ID 16: 5'-GCGGCCGCCACCGCGGTGGAGCTTTTATCCACATGGCGACAG

[0083] Specifically, as in Example 1, M. succiniciproducens PALK (KCTC10973BP) was spread on BHI solid medium and cultured at 39°C for 3 hours. Then, the colonies were inoculated into 10 mL of BHI liquid medium and cultured for 1 hour. The sufficiently grown cell culture medium was again inoculated into 1 mL of BHI liquid medium and cultured in a static incubator at 39°C. After 4 to 5 hours, cell growth was observed. 600 When the bacterial culture medium reached approximately 0.3-0.5 based on the baseline, the bacterial culture medium was left at 0°C-4°C for 2 minutes to stop cell growth, and then the cells were obtained by centrifugation at 4°C and 4,500 rpm for 15 minutes. Next, the cells were resuspended in 200 mL of 10% glycerol solution at 4°C, and then centrifuged again under the same conditions. This resuspension and centrifugation was repeated a total of three times, with the 10% glycerol solution being reduced by half each time. The finally obtained cells were resuspended in the same volume of 10% glycerol solution, aliquoted, and stored at -80°C.

[0084] The cell concentration suspension obtained above was mixed with the gene removal vector pcorAKO prepared in this example, and electroporation was performed under conditions of 2.5kV, 25μF, and 200Ω. Transformation into M. succiniciproducens PALK (KCTC10973BP) was attempted using the vector. Each electroporated cell was added to BHI liquid medium and recovered in a static incubator at 39°C for 1 hour. The entire culture medium was then spread onto BHI solid medium containing the antibiotic chloramphenicol 6.8μg / mL and cultured in a static incubator at 39°C for 48 hours or more. To select colonies that exhibited double crossover, the formed colonies were spread onto BHI solid medium containing chloramphenicol (6.8μg / mL) and 100g / L sucrose, cultured for 24 hours, and then the formed colonies were spread again onto the same solid medium.

[0085] The mutant strains formed in the aforementioned culture medium were cultured in BHI liquid medium containing antibiotics, and the genomic DNA of the cultured strains was analyzed. PCR was performed using the genomic DNA of the isolated mutant strains as a template, and the presence or absence of deletions in the corA gene of the genomic genes was confirmed by electrophoresis of the obtained PCR products.

[0086] [Example 3] Production of succinic acid using M. succinic acid strains MBEL55E (pMS3-mgtB), LPK (pMS3-mgtB), LPK7 (pMS3-mgtB), and PALFK (pMS3-mgtB).

[0087] The M. succiniciproducens MBEL55E (pMS3-mgtB), LPK (pMS3-mgtB), LPK7 (pMS3-mgtB), and PALFK (pMS3-mgtB) strains prepared in Examples 1 and 2 were cultured in 20 mL of MH5 medium (2.5 g yeast extract, 2.5 g polypeptone, 1 g NaCl, 0.02 g CaCl2·2H2O, 0.2 g MgCl2·6H2O, and 8.709 g K2HPO4 per liter) with separately sterilized glucose added as a carbon source to a concentration of 10 g / L. After incubation at 39°C for 12 hours under anaerobic conditions, the cell concentration and succinic acid production were measured. The cell concentration in the culture medium was measured using a spectrophotometer, and the cell concentration was calculated based on a calibration curve of previously measured spectrophotometer absorbance and dry cell weight. Furthermore, regarding the succinic acid concentration, samples were collected 12 hours after incubation. The collected samples were centrifuged at 13,000 rpm for 10 minutes, and the concentrations of various metabolites, succinic acid, glucose, and glycerol in the supernatant were analyzed by liquid chromatography.

[0088] [Example 4] Production of succinic acid using M. succinic acid strains PALK(pMS3-corA), PALK(pMS3-mgtA), PALK(pMS3-mgtB), PALK(pMS3-corA-mgtB), PALK(pMS3-mgtA-mgtB), PALK(pMS3-mgtA-corA), and PALKcorAKO strain

[0089] The M. succiniciproducens strains PALK(pMS3-corA), PALK(pMS3-mgtA), PALK(pMS3-mgtB), PALK(pMS3-corA-mgtB), PALK(pMS3-mgtA-mgtB), PALK(pMS3-mgtA-corA), and PALKcorAKO strains prepared in Examples 1 and 2 were cultured in 20 mL of MH5 medium (2.5 g of yeast extract, 2.5 g of polypeptone, 1 g of NaCl, 0.02 g of CaCl2·2H2O, 0.2 g of MgCl2·6H2O, and 8.709 g of K2HPO4 per liter) with separately sterilized glucose or glycerol added to a carbon source at a concentration of 10 g / L. After culturing under anaerobic conditions at 39°C for 8 hours, the cultures were transferred to 270 mL of the same medium and cultured again. Fermentation is carried out using the aforementioned culture medium in 2.5 L of synthetic medium (per liter: 1 g NaCl, 2 g (NH4)2HPO4, 0.02 g CaCl2·2H2O, 0.2 g MgCl2·6H2O, 8.709 g K2HPO4, 0.5 g cysteine, 0.5 g methionine, 0.5 g alanine, 0.5 g asparagine, 0.5 g aspartic acid, 0.5 g proline, 0.5 g serine, 0.005 g nicotinic acid) The fermentation was carried out by inoculating a microbial reactor (Bioflo 3000, New Brunswick Scientific Co., NJ, USA) containing 0.005 g of Ca-pantothenate, 0.005 g of pyridoxine·HCl, 0.005 g of thiamine, 0.005 g of ascorbic acid, and 0.005 g of biotin. The fermentation conditions were an initial glucose concentration of 18.2 g / L (100 mM), or an initial glycerol concentration of 4.6 g / L (50 mM) when glycerol was used, at a temperature of 39 °C and 200 rpm, while supplying pure carbon dioxide at a rate of 0.2 vvm (volume of carbon dioxide / working volume in the incubator / min).During fermentation, the pH was adjusted to 6.5 using 1.57 M aqueous ammonia and 6.84 M magnesium hydroxide solution, and 25 μg / mL kanamycin and 25 μg / mL ampicillin were added as antibiotics. To produce high concentrations of succinic acid, if the carbon source was completely depleted, 900 g / L glucose and glycerol solution was added semi-continuously as needed. The cell concentration in the culture medium was measured using a spectrophotometer, and the cell concentration was calculated based on a calibration curve of previously measured spectrophotometer absorbance and dry cell weight. Samples were periodically taken from the bioreactor during the fermentation process, and the collected samples were centrifuged at 13,000 rpm for 10 minutes. The concentrations of various metabolites, succinic acid, glucose, and glycerol in the supernatant were analyzed by liquid chromatography.

[0090] As a result, as shown in Figures 4, 5 and Table 5, when glucose alone was used as the carbon source, the PALK(pMS3-corA) strain yielded 86.66 g / L of succinic acid with a yield of 1.23 mol / mol relative to glucose and a productivity of 3.30 g / L / h. The PALK(pMS3-mgtA) strain yielded 82.33 g / L of succinic acid with a yield of 1.19 mol / mol relative to glucose and a productivity of 3.13 g / L / h. The PALK(pMS3-mgtB) strain yielded 94.25 g / L of succinic acid with a yield of 1.26 mol / mol relative to glucose and a productivity of 3.46 g / L / h. The PALK(pMS3-mgtA-corA) strain yielded 83.13 g / L of succinic acid with a yield of 1.26 mol / mol relative to glucose and a productivity of 3.29 g / L / h. The PALK(pMS3-corA-mgtB) strain yielded 94.12 g / L of succinic acid with a yield of 1.26 mol / mol relative to glucose and a productivity of 3.45 g / L / h. The PALK(pMS3-mgtA-mgtB) strain yielded 76.69 g / L of succinic acid with a yield of 1.19 mol / mol relative to glucose and a productivity of 3.03 g / L / h. Finally, in fed-batch fermentation of the PALKcorAKO strain, 7.05 g / L of succinic acid was obtained with a yield of 0.91 mol / mol relative to glucose and a productivity of 0.27 g / L / h. As a result, the PALK(pMS3-mgtB) strain showed the best succinic acid production ability, demonstrating improved succinic acid production compared to the conventional M. succinicin-producens PALK(KCTC10973BP) strain.

[0091] [Example 5] Improvement of succinic acid production using M. succiniciniiproducens PALK (pMS3-mgtB) strain

[0092] In this example, we confirmed a method for improving succinic acid production using the M. succiniciniiproducens PALK (pMS3-mgtB) strain.

[0093] As shown in Figure 5(b), the PALK(pMS3-mgtB) strain reached its maximum productivity 11 to 13 hours after inoculation, and it can be seen that the cell concentration was at its highest level at this time. Therefore, we first investigated how productivity changes when the cell concentration is further increased from the current level in order to maximize productivity.

[0094] Under the same culture conditions as in Example 4, the initial cell concentration of the PALK(pMS3-mgtB) strain was set to OD. 600 To investigate the change in succinic acid productivity when the concentration was increased to 20.5 (9.25 g DCW / L), the mixture was inoculated into a microbial reactor containing 2.5 L of synthetic medium. The fermentation conditions at inoculation were an initial glucose concentration of 18.2 g / L (100 mM) and, when glycerol was used, an initial glycerol concentration of 4.6 g / L (50 mM). Fermentation was carried out at a temperature of 39 °C and 200 rpm while supplying pure carbon dioxide at a rate of 0.2 vvm. The pH during fermentation was adjusted to 6.5 using 1.57 M aqueous ammonia and 6.84 M magnesium hydroxide solution, and kanamycin 25 μg / mL and chloramphenicol 6.8 μg / mL were added as antibiotics. For high-concentration succinic acid production, if the carbon source was completely depleted, 900 g / L of glucose and glycerol solution was added semi-continuously as needed. PALK(pMS3-mgtB) was fermented for 11 hours, and the culture was terminated when the cell concentration reached its peak. The culture medium was then centrifuged at 4°C and 6,000 rpm for 10 minutes to obtain a cell pellet. The cell pellet was resuspended in 200 mL of the same synthetic medium to obtain a high-concentration inoculation (inoclulum). This was inoculated and cultured under the same conditions as in Example 4, but with the initial glucose and glycerol concentrations doubled. As shown in Figure 6 and Table 5, the succinic acid productivity was 11.79 g / L / h, with a peak productivity of 40.32 g / L / h, confirming that it was more than double the productivity compared to the original fermentation conditions. This shows that the M. succinicin-producens PALK(pMS3-mgtB) strain exhibits excellent succinic acid productivity and yield when inoculated with a high concentration of cells.

[0095] [Example 6] Preparation of antibiotic markers Spectinomycin and Kanamycin deletion vectors (pSpcKO, pKmKO), mgtB gene insertion vector (pINmgtB), and preparation of the PALKmgtB strain.

[0096] For strain stability and industrial application, a pINmgtB vector was constructed by inserting the gene encoding MgtB into the genome of the M. succiniciproducens PALK (KCTC10973BP) strain. pKmKO and pSpcKO vectors were also constructed by deleting the genes encoding kanamycin and spectinomycin, antibiotic markers present in the PALK strain genome. To construct the PALK strain, a pKmKO vector deleting the kanamycin resistance gene (previously used as a selective marker for ldhA gene deletion) was created by ligating the pSacHR06 plasmid containing the sacB gene digested with XhoI and SacI, the upstream and downstream sequences of the kanamycin resistance gene (primer SEQ ID NOs. 17 and 18, SEQ ID NOs. 19 and 20), and the lox66-cat-lox77 cassette (primer SEQ ID NOs. 27 and 28) using a Gibson assembly to obtain the pKmKO. Furthermore, in order to construct a PALK strain, and to create a pSpcKO vector deleting the spectinomycin resistance gene, which had previously been used as a selection marker for pta-ackA gene deletion, we were able to obtain pSpcKO by ligating the pSacHR06 plasmid containing the sacB gene digested with XhoI and SacI, the upper and lower sequences of the spectinomycin resistance gene (primer SEQ ID NOs. 23 and 24, SEQ ID NOs. 25 and 26), and the lox66-cat-lox77 cassette (primer SEQ ID NOs. 27 and 28) using Gibson assembly. Furthermore, in order to introduce the mgtB gene into a sequence located upstream of the gene encoding CorA in the genome of the PALK strain, a linearized pSacHR06 plasmid containing the sacB gene, the upstream and downstream sequences of the gene encoding CorA (primer SEQ ID NOs. 29 and 30, SEQ ID NOs. 31 and 32), the mgtB gene from pMS3-mgtB, and the lox66-cat-lox77 cassette (primer SEQ ID NOs. 35 and 36) were ligated using Gibson assembly to obtain pINmgtB. The pSpcKO, pKmKO, and pINmgtB strains prepared as described above were introduced into M. succiniciproducens PALK (KCTC10973BP) to finally produce the PALKmgtB strain.

[0097] Sequence ID 20: 5'-ttcaacgggaaacgtcttgcTTCGGCACGCAATACGTAATC

[0098] Sequence ID 21: 5'-gtgtcacctaTACCCATATAAATCAGCATCCATG

[0099] Sequence ID 22:5'-gcctatgcggATTTGATGCTCGATGAGTTTTTC

[0100] SEQ ID NO: 23:5'-gcggccgccaccgcggtggagctACTTAGCCGGCGTATAGTTAG

[0101] Sequence ID 24:5'-tatatgggtaTAGGTGACACTATAGAACGC

[0102] Sequence ID 25: 5'-agcatcaaatCCGCATAGGCCACTAGTG

[0103] Sequence ID 26: 5'-ttcaacgggaaacgtcttgcTTCGGCACGCAATACGTAATC

[0104] Sequence ID 27: 5'-gtgtcacctaTTAATCAAATTGCTCATGATTTC

[0105] Sequence ID 28: 5'-gcctatgcggTCAATTAGGCTAATTTTATTGCAATAACAGGTGC

[0106] Sequence ID 29: 5'-gcggccgccaccgcggtggagctCCGTCTACTTCGCCGGCT

[0107] SEQ ID NO: 30: 5'-aatttgattaaTAGGTGACACTATAGAACGC

[0108] SEQ ID NO: 31: 5'-gcctaattgaCCGCATAGGCCACTAGTG

[0109] Sequence ID 32: 5'-ttcaacgggaaacgtcttgcTAATTGGGCTGATTTCGGG

[0110] Sequence ID 33: 5'-aacagaatagCTAAGGCGGCTTGCAAATG

[0111] SEQ ID NO: 34:5'-acgaacggtaAAGTATCAACTTACTTTATATGGTATC

[0112] Sequence ID 35: 5'-gcggccgccaccgcggtggagctGGAGAAAAATTTCATCACTC

[0113] Sequence ID 36: 5'-gccgccttagCTATTCTGTTGGCTAATGC

[0114] Sequence ID 37: 5'-gtgtcacctaTTAAAACCACTGGCCAAAG

[0115] Sequence ID 38: 5'-gtggttttaaTAGGTGACACTATAGAACG

[0116] Sequence ID 39: 5'-gttgatacttTACCGTTCGTATAATGTATG

[0117] Specifically, as in Example 1, M. succiniciproducens PALK (KCTC10973BP) was spread on BHI solid medium and cultured at 39°C for 3 hours. Then, the colonies were inoculated into 10 mL of BHI liquid medium and cultured for 1 hour. The sufficiently grown cell culture medium was again inoculated into 1 mL of BHI liquid medium and cultured in a static incubator at 39°C. After 4 to 5 hours, cell growth was observed. 600When the bacterial culture medium reached approximately 0.3-0.5 based on the baseline, the bacterial culture medium was left at 0°C-4°C for 2 minutes to stop cell growth, and then the cells were obtained by centrifugation at 4°C and 4,500 rpm for 15 minutes. Next, the cells were resuspended in 200 mL of 10% glycerol solution at 4°C, and then centrifuged again under the same conditions. This resuspending and centrifugation was repeated a total of three times, with the 10% glycerol solution being reduced by half each time. The finally obtained cells were resuspended in the same volume of 10% glycerol solution, aliquoted, and stored at -80°C.

[0118] The cell concentration suspension obtained above was mixed with the gene removal vector pcorAKO prepared in this example, and electroporation was performed under conditions of 2.5kV, 25μF, and 200Ω to attempt transformation of M. succiniciproducens PALK (KCTC10973BP) using the vector. BHI liquid medium was added to the electroporated cells, and recovery culture was performed for 1 hour in a static incubator at 39°C. Then, the entire culture medium was spread onto BHI solid medium containing the antibiotic chloramphenicol 6.8μg / mL, and cultured for 48 hours or more in a static incubator at 39°C. To select colonies in which double crossover occurred, the formed colonies were spread onto BHI solid medium containing chloramphenicol (6.8μg / mL) and 100g / L sucrose, cultured for 24 hours, and then the formed colonies were spread again onto the same solid medium.

[0119] The mutant strains formed in the aforementioned medium were cultured in antibiotic-containing BHI liquid medium, and the genomic DNA of the cultured strains was analyzed. PCR was performed using the genomic DNA of the isolated mutant strains as a template, and the presence or absence of insertion of the mgtB gene in the genomic genes was confirmed by electrophoresis of the obtained PCR products.

[0120] [Example 7] Production of succinic acid using M. succiniciniiproducens PALKmgtB strain

[0121] The M. succiniciproducens PALKmgtB strain prepared in Example 6 was cultured in 20 mL of MH5 medium (2.5 g yeast extract, 2.5 g polypeptone, 1 g NaCl, 0.02 g CaCl2·2H2O, 0.2 g MgCl2·6H2O, and 8.709 g K2HPO4 per liter) with separately sterilized glucose or glycerol added as a carbon source to a concentration of 10 g / L. After culturing under anaerobic conditions at 39°C for 8 hours, the culture was transferred to 270 mL of the same medium and cultured again. Fermentation is carried out in a microbial reactor (Bioflo 3000, New Brunswick Scientific) containing 2.5 L of the culture medium (per liter: 1 g NaCl, 2 g (NH4)2HPO4, 0.02 g CaCl2·2H2O, 0.2 g MgCl2·6H2O, 8.709 g K2HPO4, 0.5 g cysteine, 0.5 g methionine, 0.5 g alanine, 0.5 g asparagine, 0.5 g aspartic acid, 0.5 g proline, 0.5 g serine, 0.005 g nicotinic acid, 0.005 g Ca-pantothenic acid, 0.005 g pyridoxine·HCl, 0.005 g thiamine, 0.005 g ascorbic acid, and 0.005 g biotin) of the culture medium. The fermentation was carried out by inoculating cells in Co., NJ, USA. The fermentation conditions were as follows: an initial glucose concentration of 18.2 g / L (100 mM), and when glycerol was used, an initial glycerol concentration of 4.6 g / L (50 mM). Fermentation was carried out at a temperature of 39°C and 200 rpm while supplying pure carbon dioxide at a rate of 0.2 vvm (volume of carbon dioxide / operating volume in the culture chamber / min). The pH during fermentation was adjusted to 6.5 using 1.57 M aqueous ammonia and 6.84 M magnesium hydroxide solution. If the carbon source was completely depleted for high-concentration succinic acid production, 900 g / L glucose and glycerol solution was added semi-continuously as needed. The cell concentration in the culture medium was measured using a spectrophotometer, and the cell concentration was calculated based on a calibration curve of previously measured spectrophotometer absorbance and dry cell weight. Samples were periodically taken from the bioreactor during the fermentation process. The collected samples were centrifuged at 13,000 rpm for 10 minutes, and the concentrations of various metabolites, succinic acid, glucose, and glycerol in the supernatant were analyzed by liquid chromatography.

[0122] As a result, as shown in Figure 9, when glucose alone was used as the carbon source, the PALKmgtB strain produced 94.23 g / L of succinic acid with a yield of 1.26 mol / mol relative to glucose and a productivity of 3.45 g / L / h. Consequently, the PALKmgtB strain exhibits succinic acid production ability similar to the PALK(pMS3-mgtB) strain and demonstrates improved succinic acid production ability compared to the conventional M. succinicin-producens PALK(KCTC10973BP) strain.

[0123] [Table 1]

[0124] [Table 2]

[0125] [Table 3]

[0126] [Table 4]

[0127] [Table 5]

[0128] [Table 6] [Industrial applicability]

[0129] The succinic acid-producing mutant microorganism according to the present invention expresses a gene encoding a magnesium transporter, resulting in a significant increase in magnesium ion utilization. Therefore, when culturing anaerobic microorganisms in restriction media, it can produce high concentrations of succinic acid with the highest succinic acid productivity reported to date. Furthermore, by using advanced fermentation technology, succinic acid can be produced with even better productivity and concentration.

[0130] Although specific parts of the present invention have been described in detail above, it will be clear to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0131] JPEG2026525362000013.jpg187170 [Sequence Listing Free Text]

[0132] I attached it to an electronic file.

Claims

1. A mutant microorganism characterized by the introduction of a gene encoding a magnesium transporter into a microorganism capable of producing succinic acid.

2. The mutant microorganism according to claim 1, characterized in that the gene encoding the magnesium transporter is one or more selected from the group consisting of CorA, MgtA, and MgtB.

3. The mutant microorganism according to claim 1, characterized in that it includes the following gene encoding the magnesium transporter: (1) CorA, MgtA, or MgtB gene, (2) CorA and MgtA genes, (3) CorA and MgtB genes, or (4) MgtA and MgtB genes.

4. The mutant microorganism according to claim 2, characterized in that the CorA gene contains the sequence of sequence number 17.

5. The mutant microorganism according to claim 2, characterized in that the MgtA gene contains the sequence of sequence number 18.

6. The mutant microorganism according to claim 2, characterized in that the MgtB gene contains the sequence of Sequence ID No.

19.

7. The mutant microorganism according to claim 1, characterized in that the antibiotic resistance gene of sequence number 40 or sequence number 41 is further deleted.

8. The mutant microorganism according to claim 1, characterized in that the succinic acid-producing microorganism is selected from the group consisting of Mannheimia sp., Actinobacillus sp., Anerobiospirillum sp., Basfia sp., Escherichia coli, and Corynebacterium sp.

9. The mutant microorganism according to claim 8, characterized in that the succinic acid-producing microorganism is Mannheimia succiniciproducens.

10. The mutant microorganism according to claim 9, characterized in that the succinic acid-producing microorganism is a mutant rumen bacterium Mannheimia succiniciproduces PALK (KCTC10973BP) obtained by deleting the gene encoding pyruvate-formate hydrolytic enzyme (pfl) in Mannheimia succiniciproduces, but deleting the gene encoding lactate dehydrogenase (ldhA), the gene encoding phosphotransacetylase (pta), and the gene encoding acetate kinase (ackA).

11. The mutant microorganism according to claim 8, characterized in that the succinic acid-producing microorganism is Mannheimia succiniciproducens MBEL55E (KCTC0769BP).

12. The mutant microorganism according to claim 8, characterized in that the succinic acid-producing microorganism is Mannheimia succiniciproducts MBEL55E in which the gene encoding lactate dehydrogenase (ldhA) and the gene encoding pyruvate-formate dehydrogenase (pfl) are deleted.

13. The mutant microorganism according to claim 8, characterized in that the succinic acid-producing microorganism is a mutant strain of Mannheimia succiniciproducts LPK7 (KCTC10626BP) in which the phosphotransacetylase gene (pta) and the acetate kinase gene (ackA) are deleted.

14. The mutant microorganism according to claim 8, characterized in that the succinic acid-producing microorganism is Mannheimia succiniciproducts PALK (KCTC10973BP) in which the gene encoding fructose phosphotransferase (fruA) is deleted.

15. A method for producing succinic acid, comprising the following steps: (a) A step of culturing a mutant microorganism according to any one of claims 1 to 14 to produce succinic acid, (b) A step of recovering the succinic acid that has been produced.

16. The method for producing succinic acid according to claim 15, characterized in that the culture is carried out in a culture medium containing corn steep liquor.

17. The culture described above is Ca(OH) ２ , KOH, NaOH, Mg(OH) ２ NH ４ A method for producing succinic acid according to claim 15, characterized by adding a neutralizing agent selected from the group consisting of OH and a mixture of two or more.

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