Mutant microorganism for producing succinic acid, into which magnesium tranporter is introduced, and method for producing succinic acid using same

Abstract

The present invention relates to a mutant microorganism for producing succinic acid, into which a magnesium transporter is introduced, and a method for producing succinic acid using same and, more specifically, to a mutant microorganism for producing succinic acid, into which a gene encoding a magnesium transporter is introduced, and a method for producing succinic acid using same, wherein magnesium ion utilization is increased by using an optimized neutralizing agent.

Description

Mutant microorganism for producing succinic acid with magnesium transporter introduced therein and method for producing succinic acid using the same

[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, and more particularly, to a mutant microorganism for producing succinic acid into which a gene encoding a magnesium transporter has been introduced, characterized in that the utilization of magnesium ions is increased by using an optimized neutralizing agent, and a method for producing succinic acid using the same.

[0002]

[0003] Due to recent environmental concerns, many efforts are being made to replace the existing fossil fuel-based production of useful compounds. Accordingly, research into the production of bio-based succinic acid from renewable biomass has been conducted worldwide. Succinic acid is a four-carbon dicarboxylic acid that serves as a precursor to industrially valuable compounds such as 1,4-butanediol, γ-butyrolactone, diethyl succinate, N-methyl-2-pyrollidone, and tetrahydrofuran, and is also used as a monomer for various polymers. With the growing importance of succinic acid, numerous studies are being conducted on bio-based production methods using various microorganisms, such as Actinobacillus succinogenes, Anaerobiospirillum succiniciproducens, Escherichia coli, Mannheimia succiniciproducens, Saccharomyces cerevisiae, and Yarrowia lipolytica.

[0004] The improvement of succinic acid-producing strains was achieved by changing the metabolic flow of succinic acid-producing strains, and the strategy was to increase the flow to succinic acid by enhancing carbon metabolism and eliminating or reducing the production of by-products. That is, succinic acid production is increased by introducing or strengthening genes involved in succinic acid metabolic flow, or by deleting or weakening genes that inhibit succinic acid metabolic flow. Studies have been conducted to control the optimal pH of the culture medium by performing fed-batch fermentation with various neutralizing agents (NH4OH, Ca(OH)2, Mg(OH)2, NaOH) using these improved succinic acid-producing strains. These neutralizing agents significantly affect cell metabolism and carbon source utilization, and in particular, Mg(OH)2 was selected as the preferred neutralizing agent for succinic acid production in a previous study for the following reasons. It showed a higher carbon source utilization rate than other neutralizing agents such as Ca(OH)2, KOH, and NaOH, and the low solubility of magnesium succinate helps succinate production by reducing the osmotic pressure caused by succinate ions and the feedback inhibition effect on enzymes such as phosphoenolpyruvate carboxykinase (PCKA). In addition, Mg(OH)2 is used as a buffer to prevent acidosis in microorganisms. In particular, the magnesium ion of Mg(OH)2 itself is known to play various roles in cellular systems. Magnesium ions are involved in almost all metabolic cycles and play an important role in structural integrity and genome stability, and function as a cofactor for DNA repair proteins and an intracellular regulator of the cell cycle and apoptosis. In addition, magnesium ions act as an essential cofactor for enzymes involved in succinate production, including NADPH oxidase and PCKA. Therefore, considering the importance of magnesium ion supply, a study was conducted to increase the magnesium uptake rate by changing the magnesium transport system in E. coli to increase succinic acid production {Bioresource Technology 170(2014) 125-131}.

[0005] Under this technical background, the inventors of the present application confirmed that it is possible to develop a high-performance succinic acid-producing strain by introducing several magnesium transporters to increase magnesium ion absorption, and completed the present invention.

[0006]

[0007] Summary of the invention

[0008] The purpose of the present invention is to provide a mutant microorganism with enhanced succinic acid production ability.

[0009] The purpose of the present invention is to provide a method for maximizing succinic acid production ability using the mutant microorganism.

[0010] To achieve the above purpose, the present invention provides a mutant microorganism having a succinic acid-producing ability into which a gene encoding a magnesium transporter has been introduced.

[0011] The present invention also provides a method for producing succinic acid comprising the following steps.

[0012] (a) a step of culturing the mutant microorganism to produce succinic acid; and

[0013] (b) A step of recovering the succinic acid produced above.

[0014]

[0015] Figure 1 shows the growth and metabolite production curves of fed-batch culture of M. succiniciproducens PALK strain using glucose as a single carbon source and (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 as neutralizing agents.

[0016] Figure 2 shows the growth and metabolite production curves of fed-batch cultures of M. succiniciproducens PALK strain using glucose as a sole carbon source and using a mixture of (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 as a neutralizing agent.

[0017] Figure 3 is a two-dimensional gel image showing the physiological changes of the PALK strain of M. succiniciproducens by Mg(OH)2. It is a two-dimensional image of the whole cell proteome in (a) logarithmic phase and (b) stationary phase using NH4OH as a neutralizing agent, and a two-dimensional image of the whole cell proteome in (c) logarithmic phase and (d) stationary phase using a mixture of NH4OH and Mg(OH)2 as a neutralizing agent.

[0018] Figure 4 shows the growth and metabolite production curves of (a) PALKcorAKO, (b) PALK (pMS3-corA) strains in fed-batch culture using glucose as a sole carbon source to verify the magnesium transporter system in M. succiniciproducens. (c) is a schematic diagram of the introduction of magnesium transporters into M. succiniciproducens. (d) is a comparative graph of the intracellular magnesium ion concentrations of strains into which various magnesium transporter genes have been introduced.

[0019] Figure 5 shows the growth and metabolite production curves of M. succiniciproducens (a) PALK (pMS3-mgtA), (b) PALK (pMS3-mgtB), (c) PALK (pMS3-mgtA-corA), (d) PALK (pMS3-corA-mgtB), (e) PALK (pMS3-mgtA-mgtB) strains in fed-batch culture using glucose as a single carbon source, and (f) is a graph comparing the succinic acid concentration and productivity of the constructed strains.

[0020] Figure 6 shows the growth and metabolite production curves of the M. succiniciproducens PALK (pMS3-mgtB) strain in fed-batch culture using (a) glucose and glycerol together as carbon sources and (b) a high cell density.

[0021] Figure 7 shows the growth and metabolite production curves of fed-batch cultures of M. succiniciproducens PALK (pMS3-mgtB) strain using glucose as a sole carbon source, with fermentation medium compositions of (a) 0 CDM, (b) 1 CDM, (c) 2 CDM, (d) 0 CDM and 20 g / L corn steep liquor, (e) 1 CDM and 20 g / L corn steep liquor, and (f) 2 CDM and 20 g / L corn steep liquor.

[0022] Figure 8 is a graph showing the amino acid analysis results of corn steep liquor.

[0023] Figure 9 shows the growth and metabolite production curves of the M. succiniciproducensPALKmgtB strain in an antibiotic-free fed-batch culture using glucose as a single carbon source.

[0024] Figure 10 is a comparative graph showing the increase in growth and succinic acid production through 16-hour cultivation of M. succiniciproducens MBEL55E (pMS3-mgtB), LPK (pMS3-mgtB), LPK7 (pMS3-mgtB), and PALFK (pMS3-mgtB) strains using glucose as a single carbon source.

[0025]

[0026] Detailed description and specific implementation examples of the invention

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0028] The inventors of this application sought a method to significantly improve succinic acid productivity compared to conventional methods and investigated the effect of magnesium ions on succinic acid production. The effects of various neutralizing agents on succinic acid productivity were verified in M. succiniciproducens and the composition of neutralizing agents was optimized. Furthermore, the physiological effects of magnesium ions were analyzed and a gene acting as a magnesium transporter was discovered.

[0029] Based on this, the present invention relates to a mutant microorganism having a succinic acid-producing ability into which a gene encoding a magnesium transporter has been introduced.

[0030] 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.

[0031] Specifically, the gene encoding the magnesium transporter may include, but is not limited to, the following:

[0032] (1) CorA, MgtA or MgtB gene;

[0033] (2) CorA and MgtA genes;

[0034] (3) CorA and MgtB genes; or

[0035] (4) MgtA and MgtB genes.

[0036] In one embodiment, the CorA gene may comprise the sequence of SEQ ID NO: 17.

[0037] [Sequence number 17]

[0038]

[0039] In another embodiment, the MgtA gene may comprise the sequence of SEQ ID NO: 18.

[0040] [Sequence number 18]

[0041]

[0042] In another embodiment, the MgtB gene may comprise the sequence of SEQ ID NO: 19.

[0043] [Sequence number 19]

[0044]

[0045] The above mutant microorganism may have an additional deletion of an antibiotic resistance gene. Specifically, it may be characterized by an additional deletion of the antibiotic resistance gene of SEQ ID NO: 40 or SEQ ID NO: 41. This may enable cultivation in a medium without the addition of antibiotics.

[0046] [Sequence number 40]

[0047]

[0048] [Sequence number 41]

[0049]

[0050] In the present invention, "overexpression" refers to a level of expression higher than the level at which the corresponding gene is expressed in a cell under normal conditions, and is a concept that includes increasing the amount of expression by replacing the promoter of a gene existing in the genome with a strong promoter or cloning the corresponding gene into an expression vector and transforming the cell.

[0051] As used herein, "vector" refers to a DNA construct containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in a suitable host. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases, can integrate into the genome itself. Since plasmids are currently the most commonly used form of vector, the terms "plasmid" and "vector" are sometimes used interchangeably herein. For the purposes of the present invention, plasmid vectors are preferred. A typical plasmid vector useful for this purpose has a structure that includes (a) an origin of replication that allows efficient replication, such that hundreds of plasmid vectors can be contained per host cell, (b) an antibiotic resistance gene that allows selection of host cells transformed with the plasmid vector, and (c) a restriction enzyme cleavage site into which a foreign DNA fragment can be inserted. Even if an appropriate restriction enzyme cleavage site does not exist, the vector and foreign DNA can be easily ligated using a synthetic oligonucleotide adaptor or linker according to conventional methods.

[0052] After ligation, the vector must be transformed into a suitable host cell. The microorganism having the ability to produce succinic acid may be characterized in that the microorganism having the ability to produce succinic acid is selected from the group consisting of Mannheimia sp., Actinobacillus sp., Anaerobiospirillum sp., Basfiasp., E. coli, and Corynebacterium sp. In addition, the genus Mannheimia includes the genus Basfia, which was recently found to be substantially the same genus.

[0053] Preferably, the microorganism having the ability to produce succinic acid may be characterized as Mannheimia succiniciproducensPALK (KCTC10973BP).

[0054] In some cases, Mannheimia succiniciproducens may contain a Basfia mutant strain that contains a gene encoding a magnesium transporter, which is very similar to the Basfia strain.

[0055] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. This can be a gene and regulatory sequence(s) that are linked in such a way that an appropriate molecule (e.g., a transcriptional activating protein) enables gene expression when bound to the regulatory sequence(s). For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it influences the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it influences the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are in contact, and in the case of a secretory leader, are in contact and in reading frame. However, an enhancer need not be in contact. These sequences are joined by ligation at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used according to conventional methods.

[0056] The term "expression vector" as used herein typically refers to a recombinant carrier into which a fragment of heterologous DNA has been inserted, typically a fragment of double-stranded DNA. Here, heterologous DNA refers to heterologous DNA, which is DNA not naturally found in the host cell. Once within a host cell, the expression vector can replicate independently of the host chromosomal DNA, producing multiple copies of the vector and its inserted (heterologous) DNA.

[0057] As is well known in the art, to increase the level of expression of a transfected gene in a host cell, the gene must be operably linked to transcriptional and translational expression control sequences that function within the selected expression host. Preferably, the expression control sequences and the gene are contained within a single expression vector that also includes a bacterial selection marker and an origin of replication. If the host cell is eukaryotic, the expression vector must further include an expression marker useful in the eukaryotic expression host.

[0058] A host cell transformed or transfected with the expression vector described above constitutes another aspect of the present invention. The term "transformation" as used herein refers to the introduction of DNA into a host cell, such that the DNA becomes replicable either as an extrachromosomal element or through chromosomal integration. The term "transfection" as used herein refers to the uptake of an expression vector by a host cell, regardless of whether any coding sequence is actually expressed.

[0059] It should be understood that not all vectors and expression control sequences are equally effective in expressing the DNA sequences of the present invention. Similarly, not all hosts will function equally well in the same expression system. However, those skilled in the art can appropriately select from among various vectors, expression control sequences, and hosts without undue experimental burden or deviation from the scope of the present invention. For example, when selecting a vector, consideration should be given to the host, as the vector must replicate within it. The vector's copy number, its ability to control copy number, and the expression of other proteins encoded by the vector, such as antibiotic markers, should also be considered. When selecting an expression control sequence, various factors should be considered, such as the relative strength of the sequence, its controllability, and its compatibility with the DNA sequences of the present invention, particularly with respect to potential secondary structures. The unicellular host should be selected based on factors such as the selected vector, the toxicity of the product encoded by the DNA sequence of the present invention, secretion characteristics, the ability to accurately fold the protein, culture and fermentation requirements, and the ease of purifying the product encoded by the DNA sequence of the present invention from the host. Within the scope of these variables, one of ordinary skill in the art can select various vector / expression control sequence / host combinations capable of expressing the DNA sequence of the present invention in fermentation or large-scale animal culture.

[0060] Based on this, the present invention relates to a method for producing succinic acid, comprising, from another perspective, (a) a step of culturing the mutant microorganism to produce succinic acid; and (b) a step of recovering the produced succinic acid.

[0061] The above culture may be characterized by using glucose and / or glycerol as a carbon source.

[0062] The above cultivation may be performed under anaerobic conditions, but is not limited thereto.

[0063] The above cultivation may be characterized in that it is performed in a medium containing corn steep liquor. In some cases, it may contain a chemically defined media (CDM).

[0064] The above cultivation can be performed by adding a neutralizing agent selected from the group consisting of Ca(OH)2, KOH, NaOH, Mg(OH)2, NH4OH, and two or more thereof. The neutralizing agent significantly affects cell metabolism and carbon source utilization, and was selected as a preferred neutralizing agent for succinic acid production.

[0065] When using the above 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.

[0066] The microorganism having the above succinic acid-producing ability may be characterized in that the microorganism having the above succinic acid-producing ability is selected from the group consisting of Mannheimia sp., Actinobacillus sp., Anaerobiospirillum sp., Basfiasp., E. coli, and Corynebacterium sp. In addition, the genus Mannheimia includes the genus Basfia, which was recently found to be substantially the same genus.

[0067] The microorganism having the above succinic acid producing ability may be, for example, Mannheimia succiniciproducens.

[0068] The microorganism having the above succinic acid producing ability may be Mannheimia succiniciproducensMBEL55E (KCTC0769BP).

[0069] The present inventors have identified the strain M. succiniciproducens MBEL55E (KCTC0769BP) that produces succinic acid with high efficiency from Korean cattle, and have published the genome sequence and metabolic characteristics of the strain (Hong et al., Nature Biotechnol., 22:1275, 2004). In addition, the present inventors have created a mutant strain, M. succiniciproducens LPK (KCTC10558BP), in which the lactate dehydrogenase gene (ldhA) and the pyruvate-formate lyase gene (pfl) are deleted to suppress the production of lactic acid and formic acid in M. succiniciproducens MBEL55E, a type of lumen bacterium.

[0070] The microorganism having the above succinic acid production ability can be characterized as LPK (KCTC10558BP) in which the lactate dehydrogenase coding gene (ldhA) and the pyruvate-formate lyase coding gene (pfl) are deleted from Mannheimia succiniciproducensMBEL55E.

[0071] The microorganism having the above succinic acid-producing ability can be characterized as a mutant lumenobacterium Mannheimia succiniciproducens PALK (KCTC10973BP) obtained by deleting the gene encoding lactate dehydrogenase (ldhA), the gene encoding phosphotransacetylase (pta), and the gene encoding acetate kinase (ackA) without deleting the gene encoding pyruvate-formate lyase (pfl) in Mannheimia succiniciproducens.

[0072] The present inventors have identified a strain, M. succiniciproducens MBEL55E (KCTC0769BP), that produces succinic acid from Korean cattle with high efficiency, and have published the genome sequence and metabolic characteristics of the strain (Hong et al., Nature Biotechnol., 22:1275, 2004). In addition, the present inventors have created a mutant strain, M. succiniciproducens LPK (KCTC10558BP), in which the lactate dehydrogenase gene (ldhA) and the pyruvate-formate lyase gene (pfl) are deleted in M. succiniciproducens MBEL55E, a type of lumen bacteria, to suppress the production of lactic acid and formic acid. Furthermore, the mutant strain M. A mutant strain M. succiniciproducensLPK7 (KCTC10626BP) was created by deleting the phosphotransacetylase gene (pta) and the acetate kinase gene (ackA) from M. succiniciproducensLPK, and culturing it under anaerobic conditions produced succinic acid (International Patent Publication No. WO05 / 052135).

[0073] From the genome information of Mannheimia succiniciproducens 55E, a type of lumen bacteria, the lactate dehydrogenase gene (ldhA) and the pyruvate-formate lyase gene (pfl) were found, and then, using a gene deletion vector, both genes were deleted from the genome of Mannheimia succiniciproducens 55E to produce a mutant strain [Mannheimia sp. LPK (Mannheimia sp. LPK, KCTC 10558BP)] (Korean Patent No. 10-0556099).

[0074] The microorganism having the above succinic acid producing ability may be a mutant strain Mannheimia succiniciproducensLPK7 (KCTC10626BP) in which the phosphotransacetylase gene (pta) and the acetic acid kinase gene (ackA) are deleted from Mannheimia succiniciproducensLPK (Korean Patent No. 10-0630819).

[0075] The microorganism having the above succinic acid-producing ability may be a mutant microorganism Mannheimia succiniciproducens PALK (KCTC10973BP) that has the characteristic of producing only succinic acid at a high concentration while producing almost no other organic acids except succinic acid under anaerobic conditions, obtained by deleting the gene encoding pyruvate-formate lyase (pfl) in Mannheimia succiniciproducens and the gene encoding lactate dehydrogenase (ldhA), the gene encoding phosphotransacetylase (pta), and the gene encoding acetate kinase (ackA) (Korean Patent No. 10-0780324).

[0076] The microorganism having the above succinic acid-producing ability may be M. succiniciproducens PALFK (KCTC11694BP), a strain having the ability to metabolize sucrose and glycerol simultaneously by deleting the gene (fruA) encoding fructose phosphotransferase from the genomic DNA of Mannheimia succiniciproducens PALK (KCTC10973BP) (International Patent Publication No. WO2012030130).

[0077] In the present invention, the fermentation process was optimized to overproduce succinic acid. In particular, selecting the most appropriate neutralizing agent to maintain the optimal pH (pH 6.5) of M. succiniciproducens is important for increasing succinic acid production. Therefore, to find the most effective neutralizing agent for succinic acid production, five neutralizing agents were selected: NH4OH, KOH, Ca(OH)2, Mg(OH)2, and NaOH. M. succiniciproducens PALK (KCTC10973BP) was selected as a platform strain. The M. succiniciproducens PALK (KCTC10973BP) strain is M. This strain is a succinic acid-producing mutant microorganism obtained by deleting the genes encoding lactate dehydrogenase, phosphotransacetylase, and acetate kinase from the wild-type strain of M. succiniciproducens. It is characterized by producing only succinic acid in high concentrations under anaerobic conditions while producing almost no other organic acids. First, when 8.4 M Ca(OH)2 was used as a neutralizing agent, 16.79 g / L of succinic acid was produced in fed-batch fermentation with a yield and productivity of 0.56 mol / mol glucose and 0.67 g / L / h, respectively (Fig. 1; Table 1). However, calcium precipitate formation was observed immediately after the addition of Ca(OH)2, because calcium ions were immediately converted to calcium carbonate, a white precipitate that is insoluble in water, together with carbonic acid in the culture medium. This precipitate formation was observed in M. succiniciproducens requires a continuous supply of carbon dioxide (1 mol of carbon dioxide is processed to produce 1 mol of succinic acid), which should be avoided. Therefore, considering the low succinic acid productivity and the formation of calcium carbonate precipitates, Ca(OH)2 was confirmed to be not a suitable neutralizing agent. Next, KOH and NaOH, which are strongly alkaline, were used as neutralizing agents. Fed-batch fermentation using KOH as a neutralizing agent resulted in the production of 25.53 g / L of succinic acid with 0.93 mol / mol glucose and 0.While 88 g / L / h of succinic acid was produced with a yield and productivity, in the case of NaOH, 33.16 g / L of succinic acid was produced with a yield of 1.02 mol / mol glucose and a productivity of 1.15 g / L / h (Fig. 1; Table 1). However, when KOH or NaOH was used, cell aggregation problems were found. This is because when high concentrations of potassium and sodium ions are present in the fermentation medium, a hyperosmotic environment can be created, which can damage the morphology of the bacteria. As a result, it was revealed that when KOH or NaOH was used as a neutralizing agent, low cell growth and low succinic acid production were observed. In addition, NH4OH is a medium alkaline solution widely used in industry as a nitrogen source for microorganisms and is used to control pH during fermentation. Fed-batch fermentation using 28% w / w NH4OH as a neutralizing agent produced 58.6 g / L of succinic acid with a yield and productivity of 1.04 mol / mol glucose and 2.44 g / L / h, respectively (Fig. 1; Table 1).

[0078] 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 increases carbon utilization. Furthermore, the lower solubility of magnesium succinate compared to other succinates, such as sodium and potassium succinate, reduces feedback inhibition by the succinate anion when using Mg(OH)2. Furthermore, Mg(OH)2 has been reported to provide strong buffering capacity and inherent insolubility under overfeeding conditions. Therefore, when Mg(OH)2 is overfed, it does not cause a rapid increase in pH that can seriously impair the fermentation process. Instead, the insoluble Mg(OH)2, when combined with the nutrients and biosolids matrix present in the fermenter, provides a gradual pH increase and strong buffering capacity. Furthermore, the magnesium ion of Mg(OH)2 itself is known to play various roles in cellular systems, including structural integrity and genome stability, DNA repair, intracellular regulator of cell cycle and apoptosis, and most importantly, succinate production. However, fed-batch fermentation using only Mg(OH)2 as a neutralizing agent confirmed the formation of magnesium carbonate precipitate, and 33.72 g / L of succinate was produced with a yield and productivity of 1.04 mol / mol glucose and 1.37 g / L / h, respectively (Fig. 1; Table 1). Therefore, mixtures of NH4OH and Mg(OH)2 were tested at various concentrations (6.15 M NH4OH and 1.79 M Mg(OH)2, 4.1 M NH4OH and 3.56 M MgOH2, 3.08 M NH4OH and 5.35 M MgOH2, 1.45 M NH4OH and 7.15 M Mg(OH)2, 1.57 M NH4OH and 6.84 M Mg(OH)2, 0.96 M NH4OH and 7.85 M Mg(OH)2) to determine the optimal amount of each component (Fig. 2; Table 1). It was found that both cell growth and succinic acid production increased as the fraction of Mg(OH)2 in the mixture increased.This increase phenomenon began to level off when the concentration of Mg(OH)2 in the mixture exceeded 6.84 M, confirming a slight decrease in succinic acid productivity. In addition, compared to fermentation using only NH4OH, it was confirmed that cell concentration (35% increase) and succinic acid production (27% increase) increased significantly. Therefore, the optimized mixture (1.57 M NH4OH and 6.84 M Mg(OH)2) was used as an optimized neutralizing agent for M. succiniciproducens in the present invention.

[0079] To determine the reason for the enhanced cell growth and succinic acid production induced by Mg(OH)2 treatment, the physiological changes in the M. succiniciproducens PALK (KCTC10973BP) strain were analyzed. Whole-cell proteome samples were collected during fed-batch fermentation of M. succiniciproducens PALK (KCTC10973BP) at logarithmic growth phase (10 h after the start of fermentation) and stationary phase (16 h after the start of fermentation) (Fig. 3). The intensities of protein spots found in two-dimensional gels were determined based on the proteome reference map and over 200 proteins identified and characterized in a previous study (Lee et al., Bioprocess Biosyst Eng 2010, 33:97-107) (Fig. 3). The fold difference between the spots obtained during logarithmic and stationary phases was calculated based on the comparison of the analyzed spot intensities and areas (Table 2).

[0080] As expected, enzymes involved in succinate production, such as PckA and FumC, increased in both logarithmic and stationary phases when a mixture of NH4OH and Mg(OH)2 was used as a neutralizing agent compared to fed-batch fermentation using NH4OH alone. In addition, we found that the corresponding enzymes (PckA, fructose kinase, enolase, glyceraldehyde 3-phosphate dehydrogenase, 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase), TCA cycle enzymes (fumarate hydratase, malate dehydrogenase), sugar-binding proteins (phosphomanomutase), and phosphotransferase systems (phosphocarrier protein HPr, sugar-specific IIA component) increased. In particular, the fold change was much higher in the logarithmic phase, confirming the importance of Mg(OH)2 for stable cell growth. Furthermore, when a mixture of NH4OH and 6.84 M Mg(OH)2) was used as a neutralizing agent due to active metabolism during the logarithmic growth phase, a much higher cell density was observed.

[0081] As confirmed in the above study, the use of Mg(OH)2 as a neutralizing agent was found to be beneficial for succinic acid production, so we analyzed the intracellular magnesium ion concentration of the M. succiniciproducens PALK (KCTC10973BP) strain (Fig. 4; Table 3-4). The intracellular magnesium ion concentration was confirmed to be much higher in the PALK strain (3.452 mM) than in other strains such as Escherichia coli or Bacillus subtilis (1–2 mM). In addition, the genome sequence of M. succiniciproducens was investigated to verify the magnesium transport system in M. succiniciproducens. In a previous study (Hong et al., Nat. Biotechnol., 2004, 22:1275-1281), wild-type M. Genome analysis of the strain P. succiniciproducensMBEL55E (KCTC0769BP) revealed that this facultative anaerobic bacterium possesses the corA gene as a magnesium transporter, whereas the mgtA or mgtB genes, known as independent magnesium transporters like corA, are absent from its genome. To further investigate the importance of the corA gene, the PALKcorAKO strain, in which the corA gene was deleted from the PALK strain genome, was constructed. Fed-batch fermentation revealed that cells could hardly grow (Fig. 4). Therefore, to determine the effect of increased intracellular magnesium ion concentration on cell growth and succinate production, the corA gene was overexpressed in the PALK strain. As a result, the PALK (pMS3-corA) strain was found to uptake more magnesium ions, showing a higher intracellular magnesium ion concentration (5.814 mM) than the PALK strain (3.452 mM). As a result of fed-batch fermentation, 86.66 g / L of succinic acid was produced with a yield and productivity of 1.23 mol / mol glucose and 3.30 g / L / h, respectively (Fig. 4; Table 5).

[0082] To enhance succinic acid production by introducing a magnesium transporter, genes encoding magnesium transporters from other microorganisms were introduced into the M. succiniciproducens PALK (KCTC10973BP) strain. First, the mgtA gene, which functions as a magnesium transporter in E. coli, was introduced. The PALK (pMS3-mgtA) strain developed in this way absorbed a large amount of magnesium ions, and the intracellular magnesium ion concentration was higher (6.240 mM) than that of the PALK strain (3.452 mM), but similar to that of the PALK (pMS3-corA) strain (5.814 mM) (Fig. 4; Table 3-4). Fed-batch fermentation of the PALK (pMS3-mgtA) strain produced 82.33 g / L of succinic acid, with a yield and productivity of 1.19 mol / mol glucose and 3.13 g / L / h, respectively (Fig. 5; Table 5). Next, the mgtB gene, which functions as a magnesium transporter in Salmonella enterica, was also introduced into the PALK strain. The PALK (pMS3-mgtB) strain developed in this way absorbed a large amount of magnesium ions and had the highest intracellular magnesium ion concentration (8.474 mM) among the M. succiniciproducens strains (Fig. 4; Tables 3-4). In addition, fed-batch fermentation of the PALK (pMS3-mgtB) strain produced 94.25 g / L of succinic acid with a yield and productivity of 1.26 mol / mol glucose and 3.46 g / L / h, respectively (Fig. 5; Table 5). Furthermore, to confirm the synergistic effect of simultaneously overexpressing multiple magnesium transporters, three strains were constructed in which two different magnesium transporters were overexpressed. The results of fed-batch fermentation of the PALK (pMS3-mgtA-corA), PALK (pMS3-corA-mgtB), and PALK (pMS3-mgtA-mgtB) strains developed in this way were 83.13, 94.12, and 76.69 g / L of succinic acid, 1.26, 1.26, and 1.19 mol / mol glucose, and 3.29, 3.45, and 3.It was produced with a yield and productivity of 0.03 g / L / h (Fig. 5; Table 5). Consequently, it was confirmed that succinic acid production did not significantly increase in M. succiniciproducens strains overexpressing different magnesium transporters.

[0083] In summary, the above heterologous magnesium transporter introduction studies showed that the PALK (pMS3-mgtB) strain had significantly higher succinic acid titer, yield, and productivity than other strains. Therefore, the PALK (pMS3-mgtB) strain was used to optimize the culture conditions. To further enhance the succinic acid production of the constructed PALK (pMS3-mgtB) strain, fed-batch fermentation was performed using glucose and glycerol together as carbon sources under limited medium. It is known that the use of glycerol can be helpful in succinic acid production because glycerol provides twice the reducing equivalents of glucose on a molar basis (Ahnet et al., Nat. Commun., 2020, 11:1970). When the PALK (pMS3-mgtB) strain was fed-batch fermented using both glucose and glycerol as carbon sources under a limited medium, 110.92 g / L of succinic acid was produced with a yield and productivity of 1.42 mol / mol glucose (for ease of yield comparison, when glucose and glycerol were used together as carbon sources, the carbon numbers of both carbon sources were included in the calculation and expressed based on glucose, and hereinafter, it is consistently expressed as 'mol / mol glucose') and 4.07 g / L / h, respectively (Fig. 6; Table 5). This result confirmed a significant increase compared to the previous fed-batch fermentation results using glucose as the sole carbon source. In addition, one of the fermentation characteristics of M. succiniciproducens is that the cell density is low during fed-batch fermentation. For 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 in this study, the maximum cell densities during fermentation using glucose were 3.52, 4.19, 3.45, 3.08, 3.01, and 3.58 gDCW / L, respectively.On the other hand, the productivity per cell of these strains was 0.94, 0.75, 1.00, 1.07, 1.15, and 0.85 g / gDCWh, respectively. Since the productivity per cell of these constructed strains is very high, the productivity is very high even though the cell density is much lower than that of other succinic acid-producing strains. Since the cell itself is a cell factory, the productivity can be further increased by increasing the cell density. Therefore, the constructed PALK (pMS3-mgtB) strain was inoculated at a high concentration to produce succinic acid with improved productivity under anaerobic conditions. In a limited medium, a high initial cell concentration of 9.25 gDCW / L was inoculated, and fed-batch fermentation was performed using glucose and glycerol together as carbon sources. As a result, 153.23 g / L of succinic acid was produced with a yield and productivity of 1.25 mol / mol glucose and 11.7 g / L / h, respectively.

[0084] Furthermore, to reduce production costs, the fermentation culture conditions of the constructed strain were optimized. To lower production costs, we removed these supplements from the current fermentation medium, which contained seven amino acids and six vitamins developed in a previous 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. succiniciproducens, the PALK (pMS3-mgtB) strain was cultured in a limited medium without amino acids or vitamins. As a result, the PALK (pMS3-mgtB) strain was unable to grow or produce succinic acid without supplements (Fig. 7). Therefore, instead of supplying purified amino acids and vitamins, we explored other inexpensive supplements. Among the various supplements, corn steep liquor (CSL) was selected as an alternative supplement. CSL, a byproduct of the corn wet milling industry, is an excellent nitrogen source for most microorganisms because it contains significant amounts of amino acids, polypeptides, and B-complex vitamins. Before adding CSL to the fermentation medium, its amino acid composition was analyzed, and the results confirmed that CSL contained the necessary amino acids (Fig. 8). Furthermore, based on previous studies (Yue et al., Biochem. Eng. J., 2008, 39:496-502), it was confirmed that CSL contained all the necessary vitamins. Fed-batch fermentation of the PALK (pMS3-mgtB) strain supplied with CSL produced 41 g / L of succinic acid, with a yield and productivity of 1.25 mol / mol glucose and 1.61 g / L / h, respectively (Fig. 7; Table 6). In addition, cell growth was fully restored, similar to fed-batch fermentation with seven amino acids and six vitamins (Fig. 7; Table 6). In addition, cysteine ​​and nicotinic acid, which are representative amino acids and vitamins contained in the restricted medium, were additionally supplied to the fermentation medium to verify whether the supply of CSL was sufficient to replace amino acids and vitamins.As a result, fed-batch fermentation using a medium supplemented with CSL, cysteine, and nicotinic acid did not show a significant increase in cell growth or succinic acid production, indicating that CSL can replace expensive amino acids and vitamins (Fig. 7; Table 6). Consequently, while the use of CSL completely restored cell growth, the final succinic acid production concentration was only 43.5% of that of the culture supplemented with purified amino acids and vitamins.

[0085] Furthermore, the stability of metabolically engineered strains is of considerable importance in industrial applications. Strain stability is particularly important when using plasmid-based expression systems. Therefore, the development of antibiotic-free strains for succinic acid production is crucial (Choi et al., Metab. Eng., 2018, 47:463-474). To address the plasmid instability issue, a marker gene deletion strategy was used to insert the mgtB gene into the chromosome of M. succiniciproducens, eliminating antibiotic dependence for plasmid stability through antibiotic marker gene deletion (Kimet et al., FEMS Microbiol. Lett., 2008, 278;78-85). As a result, fed-batch fermentation of the final PALKmgtB strain without antibiotic addition produced 94.23 g / L of succinic acid, with a yield and productivity of 1.26 mol / mol glucose and 3.45 g / L / h, respectively (Figure 9). Therefore, the succinic acid production index of the antibiotic-free PALKmgtB strain was similar to that of the plasmid-based strain PALK (pMS3-mgtB) (Table 5), suggesting the possibility of industrial-scale succinic acid production. Furthermore, the magnesium transporter mgtB gene was also introduced into M. succiniciproducens MBEL55E (KCTC0769BP), LPK (KCTC10558BP), LPK7 (KCTC10626BP), and PALFK (KCTC11694BP), which are microorganisms with established succinic acid production capabilities. As a result of tube culture of the MBEL55E (pMS3-mgtB), LPK (pMS3-mgtB), LPK7 (pMS3-mgtB), and PALFK (pMS3-mgtB) strains developed in this way, 6.00, 7.32, 6.64, and 6.70 g / L of succinic acid were produced, respectively, for 16 hours (Fig. 10).

[0086]

[0087] Example

[0088] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0089]

[0090] In particular, in the examples below, only a microorganism of the genus Mannheimia, which is a succinic acid-producing microorganism, is exemplified as a host cell for replacing or overexpressing the gene according to the present invention. However, it will be apparent to those skilled in the art that a mutant microorganism having a succinic acid-producing ability similar to that of the present invention can be obtained by using other types of succinic acid-producing microorganisms.

[0091]

[0092] [Example 1] Construction of CorA, MgtA, and MgtB overexpression vectors (pMS3-corA, pMS3-mgtA, pMS3-mgtB, pMS3-corA-mgtB, pMS3-mgtA-mgtB, pMS3-mgtA-corA) and construction 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

[0093] To overexpress genes encoding magnesium transporters in various microorganisms, respective overexpression vectors were constructed. PCR was performed using primers of SEQ ID NOs: 1 and 2, 3 and 4, and 5 and 6, respectively, with genomic DNA from M. succiniciproducens, E. coli, and S. enterica as templates. The obtained PCR products were digested with EcoRI and KpnI restriction enzymes and cloned into the EcoRI and KpnI sites of pMS3 (Jang et al., Appl. Environ. Microb. 73(17): 5411-5420., 2007), completing the overexpression vectors pMS3-corA and pMS3-mgtA. In the case of the mgtB gene, it was digested with KpnI and PstI restriction enzymes and cloned into the KpnI and PstI sites of pMS3, completing the overexpression vector pMS3-mgtB. To simultaneously overexpress two magnesium transporters, respective overexpression vectors were constructed. PCR was performed using the pMS3-mgtB vector as a template using primers of SEQ ID NOs: 7 and 8. The obtained PCR products were digested with HindIII restriction enzyme and cloned into the HindIII site of pMS3-corA and pMS3-mgtA, respectively, to complete the overexpression vectors pMS3-corA-mgtB and pMS3-mgtA-mgtB. In addition, PCR was performed using pMS3-corA as a template using primers of SEQ ID NOs: 9 and 10. The obtained PCR products were digested with HindIII restriction enzyme and cloned into the HindIII site of pMS3-mgtA, respectively, to construct the overexpression vector pMS3-mgtA-corA.

[0094] Sequence number 1: 5'-TACAACTCTACTGGGGAGGATGATAAATGCATTTGCAC

[0095] Sequence number 2: 5'-TCTAGAGGATCCCCGGGTACTTACAACCAGCCTTTACG

[0096] Sequence number 3: 5'-ACTTTTATCAACTCTACTGGGGAGGATGTTTAAAGAAATTTTTAC

[0097] Sequence number 4: 5'-TCTAGAGGATCCCCGGGTACTTATTGCCAGCCGTAACGAC

[0098] Sequence number 5: 5'-GAGGAATTCGAGCTCGGTACATGACTGACATGAACATTG

[0099] SEQ ID NO: 6: 5'-GCCAAGCTTGCATGCCTGCATTAAAACCACTGGCCAAAG

[0100] SEQ ID NO: 7: 5'-AGTCGACCTGCAGGCATGCACTATTCTGTTGGCTAATGC

[0101] SEQ ID NO: 8: 5'-GTCGCCCGCCAAAACAGCCATTAAAACCACTGGCCAAAG

[0102] SEQ ID NO: 9: 5'-AGTCGACCTGCAGGCATGCACTATTCTGTTGGCTAATGC

[0103] SEQ ID NO: 10: 5'-GTCGCCCGCCAAAACAGCCAAGCTTTTACAACCAGCCTTTACG

[0104]

[0105] By introducing the pMS3-corA, pMS3-mgtA, pMS3-mgtB, pMS3-corA-mgtB, pMS3-mgtA-mgtB, and pMS3-mgtA-corA constructed as above into M. succiniciproducens PALK (KCTC10973BP), the PALK (pMS3-corA), PALK (pMS3-mgtA), PALK (pMS3-mgtB), PALK (pMS3-corA-mgtB), PALK (pMS3-mgtA-mgtB), and PALK (pMS3-mgtA-corA) strains were completed, respectively. In addition, pMS3-mgtB was introduced into M. succiniciproducensMBEL55E (KCTC0769BP), LPK (KCTC10558BP), LPK7 (KCTC10626BP), PALFK (KCTC11694BP), thereby completing the MBEL55E (pMS3-mgtB), LPK (pMS3-mgtB), LPK7 (pMS3-mgtB), and PALFK (pMS3-mgtB) strains, respectively. In order to introduce the overexpression vector as described above, M. succiniciproducens PALK (KCTC10973BP), MBEL55E (KCTC0769BP), LPK (KCTC10558BP), LPK7 (KCTC10626BP), PALFK (KCTC11694BP) were plated on BHI (Brain-Heart Infusion) solid medium and cultured at 39°C for 36 hours. The colonies were inoculated into 10 mL of BHI liquid medium and cultured for 12 hours. 1 mL of the fully grown cell culture was inoculated into 100 mL of BHI liquid medium and cultured in a stationary incubator at 39°C. After about 4 to 5 hours, cell growth reached OD 600When the standard reached 0.3 to 0.5, the bacterial culture was kept at 0 to 4 ° C for 20 minutes to prevent further cell growth, and centrifuged at 4 ° C and 4,500 rpm for 15 minutes to obtain cells. Then, the cells were resuspended in 200 mL of a 10% glycerol solution at 4 ° C and centrifuged again under the same conditions as above. This resuspension and centrifugation were performed a total of 3 times while reducing the 10% glycerol solution by half. The finally obtained cells were resuspended in the same volume of a 10% glycerol solution, dispensed, and stored at -80 ° C. The obtained concentrated cell suspension and the four types of overexpression vectors produced in this example were each mixed, and electroporation was performed under the conditions of 2.5 kV, 25 μF, and 200 Ω to obtain M. Transformation of B. succiniciproducens PALK (KCTC10973BP), MBEL55E (KCTC0769BP), LPK (KCTC10558BP), LPK7 (KCTC10626BP), and PALFK (KCTC11694BP) was attempted. The electroporated cells were added to BHI liquid medium and cultured for 1 hour in a 39℃ stationary incubator. Then, the culture was spread on BHI solid medium containing 2.5 μg / mL of the antibiotic ampicillin and cultured for more than 48 hours in a 39℃ stationary incubator. The mutant strains formed in the medium were cultured in BHI liquid medium containing antibiotics, and the introduction of the overexpression vector was confirmed by electrophoresis of the vector obtained using vector mini-prep.

[0106]

[0107] [Example 2] Construction of CorA deletion vector (pcorAKO) and construction of PALKcorAKO strain

[0108] To determine whether the corA gene encoding a magnesium transporter naturally present in the M. succiniciproducens strain affects the enhancement of succinic acid production, a pcorAKO vector was constructed, which deletes the gene encoding CorA present in the genome of the M. succiniciproducens PALK (KCTC10973BP) strain. To construct pcorAKO, pSacHR06 containing the sacB gene was digested with XhoI and SacI. Then, the front and back sequences of the CorA encoding gene present in the genome were subjected to PCR using the genome of the M. succiniciproducens PALK (KCTC10973BP) strain as a template and the primers of SEQ ID NOs: 11 and 12, 13 and 14, respectively. The lox66-cat-lox77 cassette was obtained by PCR using a vector containing a chloramphenicol resistance gene as a template and the primers of SEQ ID NOs: 15 and 16. The linear pSacHR06 obtained in this way, the 1 kb each of the front and back sequences of the CorA encoding gene, and the lox66-cat-lox77 cassette were combined through Gibson assembly (Gibson et al., Nat. Methods, 6(5):343, 2009) to obtain pcorAKO. The pcorAKO constructed as above was introduced into M. succiniciproducensPALK (KCTC10973BP), thereby finally constructing the PALKcorAKO strain.

[0109] SEQ ID NO: 11: 5'-TTCAACGGGAAACGTCTTGCATGGCTGCAGTTCTTCGG

[0110] SEQ ID NO: 12: 5'-GTGTCACCTAATTTTGCTTTTAAGCTAAAAGTGCG

[0111] SEQ ID NO: 13: 5'-AAAGCAAAATAGGTGACACTATAGAACGC

[0112] SEQ ID NO: 14: 5'-AGTATGACAACCGCATAGGCCACTAGTG

[0113] SEQ ID NO: 15: 5'-GCCTATGCGGTTGTCATACTCCCTAGGTTC

[0114] SEQ ID NO: 16: 5'-GCGGCCGCCACCGCGGTGGAGCTTTTATCCACATGGCGACAG

[0115]

[0116] That is, similarly to Example 1, M. succiniciproducens PALK (KCTC10973BP) was spread on BHI solid medium and cultured at 39°C for 3 hours, and then the colonies were inoculated into 10 mL of BHI liquid medium and cultured for 1 hour. 1 mL of the sufficiently grown cell culture was inoculated again into 100 mL of BHI liquid medium and cultured in a stationary incubator at 39°C. After 4 to 5 hours, cell growth reached OD 600 When the standard reached 0.3 to 0.5, the bacterial culture was kept at 0 to 4 ° C for 2 minutes to prevent further cell growth, and centrifuged at 4 ° C and 4,500 rpm for 15 minutes to obtain cells. Then, the cells were resuspended in 200 mL of a 10% glycerol solution at 4 ° C and centrifuged again under the same conditions as above. This resuspension and centrifugation were performed a total of 3 times while reducing the 10% glycerol solution by half. The final obtained cells were resuspended in the same volume of a 10% glycerol solution, dispensed, and stored at -80 ° C.

[0117] The obtained cell concentration suspension and the gene deletion vector pcorAKO constructed in this example were mixed, and electroporation was performed under the conditions of 2.5 kV, 25 μF, and 200 Ω to attempt transformation of M. succiniciproducens PALK (KCTC10973BP) with the vector. The electroporated cells were added to BHI liquid medium and cultured for 1 hour in a 39°C stationary incubator, and then the entire culture was spread on BHI solid medium containing 6.8 μg / mL of the antibiotic chloramphenicol, and cultured for more than 48 hours in a 39°C stationary incubator. To select colonies in which double crossover had occurred, the formed colonies were plated on BHI solid medium containing chloramphenicol (6.8 μg / mL) and 100 g / L sucrose, and then cultured for 24 hours. The formed colonies were then plated again on the same solid medium.

[0118] The mutant strains formed in the above 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 obtained PCR products were subjected to electrophoresis to confirm whether the corA gene in the genomic genes was deleted.

[0119]

[0120] [Example 3] Production of succinic acid using M. succiniciproducens MBEL55E (pMS3-mgtB), LPK (pMS3-mgtB), LPK7 (pMS3-mgtB), and PALFK (pMS3-mgtB) strains

[0121] The M. succiniciproducens MBEL55E (pMS3-mgtB), LPK (pMS3-mgtB), LPK7 (pMS3-mgtB), and PALFK (pMS3-mgtB) strains produced in the above Examples 1 and 2 were inoculated into 20 mL MH5 medium (per liter, 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) with separately sterilized glucose as a carbon source at a concentration of 10 g / L, and cultured at 39°C for 12 hours under anaerobic conditions. After that, 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 using the previously measured absorbance of the spectrophotometer and the dry cell weight verification line. In addition, for the succinic acid concentration, a sample was collected after 12 hours of culture, and the collected sample was centrifuged at 13,000 rpm for 10 minutes. The various metabolites and succinic acid concentrations in the supernatant, as well as the glucose and glycerol concentrations, were analyzed by liquid chromatography.

[0122]

[0123] [Example 4] Production of succinic acid using M. succiniciproducens PALK (pMS3-corA), PALK (pMS3-mgtA), PALK (pMS3-mgtB), PALK (pMS3-corA-mgtB), PALK (pMS3-mgtA-mgtB), PALK (pMS3-mgtA-corA) and PALKcorAKO strains

[0124] M. succiniciproducens PALK (pMS3-corA), PALK (pMS3-mgtA), PALK (pMS3-mgtB), PALK (pMS3-corA-mgtB), PALK (pMS3-mgtA-mgtB), PALK (pMS3-mgtA-corA) and PALKcorAKO strains produced in Examples 1 and 2 were inoculated into 20 mL MH5 medium (per liter, 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) with separately sterilized glucose or glycerol as a carbon source at a concentration of 10 g / L, cultured under anaerobic conditions at 39°C for 8 hours, and then transferred to 270 mL of the same medium and cultured. Fermentation was performed in a microbial reactor (Bioflo 3000, New) containing 2.5 L of synthetic medium (per L, 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-pantothenate, 0.005 g pyridoxine·HCl, 0.005 g thiamine, 0.005 g ascorbic acid and 0.005 g biotin). The fermentation was performed by inoculating the medium (Brunswick Scientific Co., NJ, USA), and the fermentation conditions were 18.2 g / L (100 mM) initial glucose concentration, 4.6 g / L (50 mM) initial glycerol concentration when glycerol was used, and 0.Fermentation was performed while supplying pure carbon dioxide at a rate of 2 vvm (volume of carbon dioxide / working volume in the culture medium / min). The pH during fermentation was adjusted to 6.5 using 1.57 M ammonia water and 6.84 M magnesium hydroxide solution, and 25 μg / mL of kanamycin and 25 μg / mL of ampicillin were added as antibiotics. When the carbon source was completely consumed for the production of high-concentration succinic acid, 900 g / L of glucose and glycerol solution was added semi-continuously when necessary. The cell concentration in the culture medium was measured using a spectrophotometer, and the cell concentration was calculated using the previously measured absorbance of the spectrophotometer and the verification curve of the dry cell weight. Samples were periodically collected 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 and succinic acid, as well as glucose and glycerol in the supernatant were analyzed using liquid chromatography.

[0125] As a result, as shown in Figs. 4 and 5 and Table 5, when only glucose was used as a carbon source, the PALK (pMS3-corA) strain produced 86.66 g / L succinic acid with a yield and productivity of 1.23 mol / mol glucose and 3.30 g / L / h, respectively. The PALK (pMS3-mgtA) strain produced 82.33 g / L succinic acid with 1.19 mol / mol glucose and 3.13 g / L / h, respectively. The PALK (pMS3-mgtB) strain produced 94.25 g / L succinic acid with 1.26 mol / mol glucose and 3.46 g / L / h, respectively. The PALK (pMS3-mgtA-corA) strain produced 83.13 g / L succinic acid with a yield and productivity of 1.26 mol / mol glucose and 3.29 g / L / h, respectively. The PALK (pMS3-corA-mgtB) strain produced 94.12 g / L succinic acid with a yield and productivity of 1.26 mol / mol glucose and 3.45 g / L / h, respectively. The PALK (pMS3-mgtA-mgtB) strain produced 76.69 g / L succinic acid with a yield and productivity of 1.19 mol / mol glucose and 3.03 g / L / h, respectively. Finally, the fed-batch fermentation of the PALKcorAKO strain produced 7.05 g / L succinic acid with a yield and productivity of 0.91 mol / mol glucose and 0.27 g / L / h, respectively. As a result, the PALK (pMS3-mgtB) strain showed the best succinic acid production ability, and it shows improved succinic acid production ability compared to the existing M. succiniciproducens PALK (KCTC10973BP) strain.

[0126]

[0127] [Example 5] Enhancement of succinic acid productivity using M. succiniciproducens PALK (pMS3-mgtB) strain

[0128] In this example, a method for improving succinic acid productivity using the M. succiniciproducensPALK (pMS3-mgtB) strain was confirmed.

[0129] As shown in Figure 5(b), the PALK (pMS3-mgtB) strain reaches maximum productivity 11-13 hours after inoculation, at which point the cell concentration reaches its maximum. Therefore, to maximize productivity, we first examined how productivity changes when the cell concentration is increased above the current level.

[0130] In the same culture conditions as in Example 4, the initial cell concentration of the PALK (pMS3-mgtB) strain was OD 600= To examine how the succinic acid productivity changes when the concentration of succinic acid is increased to 20.5 (9.25 gDCW / L), the culture was inoculated into a microbial reactor containing 2.5 L of synthetic medium and the fermentation was performed under the following conditions: initial glucose concentration of 18.2 g / L (100 mM), initial glycerol concentration of 4.6 g / L (50 mM) when glycerol was used, at 39 ℃, 200 rpm, and pure carbon dioxide was supplied at a rate of 0.2 vvm. The pH during fermentation was adjusted to 6.5 using 1.57 M ammonia water and 6.84 M magnesium hydroxide solution, and antibiotics such as 25 μg / mL of kanamycin and 6.8 μg / mL of chloramphenicol were added. When the carbon source was completely consumed for high-concentration succinic acid production, 900 g / L glucose and glycerol solution was added semi-continuously if necessary. PALK (pMS3-mgtB) was fermented for 11 hours, and the culture was terminated when the cell concentration almost reached the peak, and the culture solution was centrifuged at 6,000 rpm at 4°C 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. This was inoculated and cultured under the same conditions as Example 4 but with twice the initial glucose and glycerol concentrations. As a result, as shown in Fig. 6 and Table 5, the succinic acid productivity was 11.79 g / L / h and the maximum productivity was 40.32 g / L / h, which was more than twice that of the original fermentation conditions. Through this, it can be seen that the M. succiniciproducensPALK (pMS3-mgtB) strain showed excellent succinic acid productivity and production through a fermentation method inoculating high concentrations of cells.

[0131]

[0132] [Example 6] Construction of antibiotic marker Spectinomycin, Kanamycin deletion vector (pSpcKO, pKmKO), mgtB gene insertion vector (pINmgtB), and construction of PALKmgtB strain

[0133] For strain stability and industrial application, the pINmgtB vector was constructed by inserting the gene encoding MgtB into the genome of the M. succiniciproducens PALK (KCTC10973BP) strain, and the pKmKO and pSpcKO vectors were constructed by deleting the genes encoding the antibiotic markers Kanamycin and Spectinomycin present in the PALK strain genome. To construct a pKmKO vector deleting the kanamycin resistance gene, which was previously used as a selection marker for ldhA gene deletion for PALK strain construction, the pSacHR06 plasmid containing the sacB gene digested with XhoI and SacI, the forward and reverse sequences of the kanamycin resistance gene (primer sequence numbers 17 and 18, 19 and 20), and the lox66-cat-lox77 cassette (primer sequence numbers 27 and 28) were combined by Gibson assembly to obtain pKmKO. In addition, to construct a pSpcKO vector deleting the Spectinomycin resistance gene, which was previously used as a selection marker for pta-ackA gene deletion for PALK strain construction, the pSacHR06 plasmid containing the sacB gene digested with XhoI and SacI, the front and back sequences of the Spectinomycin resistance gene (primer sequence numbers 23 and 24, 25 and 26), and the lox66-cat-lox77 cassette (primer sequence numbers 27 and 28) were combined through Gibson assembly to obtain pSpcKO.In addition, in order to insert the mgtB gene into the sequence located in front of the gene encoding CorA in the PALK strain genome, the linearized pSacHR06 plasmid containing the sacB gene, the front and back sequences of the gene encoding CorA (primer sequence numbers 29 and 30, 31 and 32), the mgtB gene from pMS3-mgtB, and the lox66-cat-lox77 cassette (primer sequence numbers 35 and 36) were combined through Gibson assembly to obtain pINmgtB. The pSpcKO, pKmKO, and pINmgtB constructed as above were introduced into M. succiniciproducens PALK (KCTC10973BP), thereby finally constructing the PALKmgtB strain.

[0134] SEQ ID NO: 20: 5'- ttcaacgggaaacgtcttgcTTCGGCACGCAATACGTAATC

[0135] SEQ ID NO: 21: 5'- gtgtcacctaTACCCATATAAATCAGCATCCATG

[0136] SEQ ID NO: 22: 5'- gcctatgcggATTTGATGCTCGATGAGTTTTTC

[0137] SEQ ID NO: 23: 5'- gcggccgccaccgcggtggagctACTTAGCCGGCGTATAGTTAG

[0138] SEQ ID NO: 24: 5'- tatatgggtaTAGGTGACACTATAGAACGC

[0139] SEQ ID NO: 25: 5'- agcatcaaatCCGCATAGGCCACTAGTG

[0140] SEQ ID NO: 26: 5'- ttcaacgggaaacgtcttgcTTCGGCACGCAATACGTAATC

[0141] SEQ ID NO: 27: 5'- gtgtcacctaTTAATCAAATTGCTCATGATTTC

[0142] SEQ ID NO: 28: 5'- gcctatgcggTCAATTAGGCTAATTTTATTGCAATAACAGGTGC

[0143] SEQ ID NO: 29: 5'- gcggccgccaccgcggtggagctCCGTCTACTTCGCCGGCT

[0144] SEQ ID NO: 30: 5'- aatttgattaaTAGGTGACACTATAGAACGC

[0145] SEQ ID NO: 31: 5'- gcctaattgaCCGCATAGGCCACTAGTG

[0146] SEQ ID NO: 32: 5'- ttcaacgggaaacgtcttgcTAATTGGGCTGATTTCGGG

[0147] SEQ ID NO: 33: 5'- aacagaatagCTAAGGCGGCTTGCAAATG

[0148] SEQ ID NO: 34: 5'- acgaacggtaAAGTATCAACTTACTTTATATGGTATC

[0149] SEQ ID NO: 35: 5'- gcggccgccaccgcggtggagctGGAGAAAAATTTCATCACTC

[0150] SEQ ID NO: 36: 5'- gccgccttagCTATTCTGTTGGCTAATGC

[0151] SEQ ID NO: 37: 5'- gtgtcacctaTTAAAACCACTGGCCAAAG

[0152] SEQ ID NO: 38: 5'- gtggttttaaTAGGTGACACTATAGAACG

[0153] SEQ ID NO: 39: 5'- gttgatacttTACCGTTCGTATAATGTATG

[0154]

[0155] That is, similarly to Example 1, M. succiniciproducens PALK (KCTC10973BP) was spread on BHI solid medium and cultured at 39°C for 3 hours, and then the colonies were inoculated into 10 mL of BHI liquid medium and cultured for 1 hour. 1 mL of the sufficiently grown cell culture was inoculated again into 100 mL of BHI liquid medium and cultured in a stationary incubator at 39°C. After 4 to 5 hours, cell growth reached OD 600 When the standard reached 0.3 to 0.5, the bacterial culture was kept at 0 to 4 ° C for 2 minutes to prevent further cell growth, and centrifuged at 4 ° C and 4,500 rpm for 15 minutes to obtain cells. Then, the cells were resuspended in 200 mL of a 10% glycerol solution at 4 ° C and centrifuged again under the same conditions as above. This resuspension and centrifugation were performed a total of 3 times while reducing the 10% glycerol solution by half. The final obtained cells were resuspended in the same volume of a 10% glycerol solution, dispensed, and stored at -80 ° C.

[0156] The obtained cell concentration suspension and the gene deletion vector pcorAKO constructed in this example were mixed, and electroporation was performed under the conditions of 2.5 kV, 25 μF, and 200 Ω to attempt transformation of M. succiniciproducens PALK (KCTC10973BP) with the vector. The electroporated cells were added to BHI liquid medium and cultured for 1 hour in a 39°C stationary incubator, and then the entire culture was spread on BHI solid medium containing 6.8 μg / mL of the antibiotic chloramphenicol, and cultured for more than 48 hours in a 39°C stationary incubator. To select colonies in which double crossover had occurred, the formed colonies were plated on BHI solid medium containing chloramphenicol (6.8 μg / mL) and 100 g / L sucrose, and then cultured for 24 hours. The formed colonies were then plated again on the same solid medium.

[0157] The mutant strains formed in the above 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 obtained PCR products were subjected to electrophoresis to confirm whether the mgtB gene was inserted into the genomic genes.

[0158]

[0159] [Example 7] Production of succinic acid using M. succiniciproducens PALKmgtB strain

[0160] The M. succiniciproducens PALKmgtB strain produced in Example 6 was inoculated into 20 mL MH5 medium (per liter, 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) with separately sterilized glucose or glycerol as a carbon source at a concentration of 10 g / L, cultured under anaerobic conditions at 39°C for 8 hours, and then transferred to 270 mL of the same medium and cultured. Fermentation was performed in a microbial reactor (Bioflo 3000, New) containing 2.5 L of synthetic medium (per L, 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-pantothenate, 0.005 g pyridoxine·HCl, 0.005 g thiamine, 0.005 g ascorbic acid and 0.005 g biotin). The fermentation was performed by inoculating the medium (Brunswick Scientific Co., NJ, USA) with an initial glucose concentration of 18.2 g / L (100 mM) and 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). The pH during fermentation was adjusted to 6.5 using 1.57 M ammonia water and 6.84 M magnesium hydroxide solution.When the carbon source was completely consumed for the production of high concentrations of succinic acid, 900 g / L glucose and glycerol solutions were added semi-continuously as needed. The cell concentration in the culture medium was measured using a spectrophotometer, and the cell concentration was calculated using the previously measured absorbance of the spectrophotometer and the dry cell weight verification line. Samples were periodically collected from the bioreactor during the fermentation process, and the collected samples were centrifuged at 13,000 rpm for 10 minutes, and the various metabolites and succinic acid concentrations, glucose, and glycerol concentrations in the supernatant were analyzed by liquid chromatography.

[0161] As a result, as shown in Fig. 9, when only glucose was used as a carbon source, the PALKmgtB strain produced 94.23 g / L succinic acid with a yield and productivity of 1.26 mol / mol glucose and 3.45 g / L / h, respectively. Consequently, the PALKmgtB strain showed similar succinic acid production ability to the PALK (pMS3-mgtB) strain, and showed improved succinic acid production ability compared to the existing M. succiniciproducens PALK (KCTC10973BP) strain.

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170] The succinic acid-producing mutant microorganism according to the present invention expresses a gene encoding a magnesium transporter, thereby significantly increasing magnesium ion utilization. Therefore, when culturing anaerobic microorganisms under a limited medium, it can produce high concentrations of succinic acid with the highest succinic acid productivity reported to date. Furthermore, by utilizing advanced fermentation technology, succinic acid can be produced with even greater productivity and concentration.

[0171]

[0172] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0173]

[0174]

[0175] Electronic file attached.

Claims

1. A mutant microorganism into which a gene encoding a magnesium transporter has been introduced into a microorganism capable of producing succinic acid.

2. A mutant microorganism in the first paragraph, wherein the gene encoding the magnesium transporter is at least one selected from the group consisting of CorA, MgtA, and MgtB.

3. In the first paragraph, a mutant microorganism characterized in that the gene encoding the magnesium transporter comprises: (1) CorA, MgtA or MgtB gene; (2) CorA and MgtA genes; (3) CorA and MgtB genes; or (4) MgtA and MgtB genes.

4. In the second paragraph, the CorA gene is a mutant microorganism comprising the sequence of SEQ ID NO:

17.

5. In the second paragraph, the MgtA gene is a mutant microorganism comprising the sequence of SEQ ID NO:

18.

6. In the second paragraph, the MgtB gene is a mutant microorganism comprising the sequence of SEQ ID NO:

19.

7. A mutant microorganism characterized in that the antibiotic resistance gene of sequence number 40 or sequence number 41 is additionally deleted in the first paragraph.

8. A mutant microorganism according to claim 1, characterized in that the microorganism having the ability to produce succinic acid is selected from the group consisting of Mannheimia sp., Actinobacillus sp., Anaerobiospirillum sp., Basfiasp., E. coli, and Corynebacterium sp.

9. A mutant microorganism according to claim 8, characterized in that the microorganism having the ability to produce succinic acid is Mannheimia succiniciproducens.

10. In the 9th paragraph, the microorganism having the ability to produce succinic acid is a mutant lumen bacterium Mannheimia succiniciproducens PALK (KCTC10973BP) obtained by deleting the lactate dehydrogenase coding gene (ldhA), the phosphotransacetylase coding gene (pta), and the acetic acid kinase coding gene (ackA) without deleting the pyruvate-formate lyase coding gene (pfl) in Mannheimia succiniciproducens.

11. A mutant microorganism according to claim 8, characterized in that the microorganism having the ability to produce succinic acid is Mannheimia succiniciproducensMBEL55E (KCTC0769BP).

12. In the 8th paragraph, the microorganism having the ability to produce succinic acid is a mutant microorganism characterized in that it is LPK (KCTC10558BP) in which the lactate dehydrogenase coding gene (ldhA) and the pyruvate-formate lyase coding gene (pfl) are deleted from Mannheimia succiniciproducensMBEL55E.

13. In the 8th paragraph, the microorganism having the ability to produce succinic acid is a mutant microorganism, characterized in that it is a mutant strain Mannheimia succiniciproducensLPK7 (KCTC10626BP) in which the phosphotransacetylase gene (pta) and the acetic acid kinase gene (ackA) are deleted from Mannheimia succiniciproducensLPK.

14. In the 8th paragraph, the microorganism having the ability to produce succinic acid is a mutant microorganism characterized in that it is Mannheimia succiniciproducens PALFK (KCTC11694BP) in which the gene (fruA) encoding fructose phosphotransferase is deleted from Mannheimia succiniciproducens PALK (KCTC10973BP).

15. A method for producing succinic acid comprising the following steps: (a) a step of culturing a mutant microorganism of any one of claims 1 to 14 to produce succinic acid; and (b) A step of recovering the succinic acid produced above.

16. A method for producing succinic acid, characterized in that the culturing is performed in a medium containing corn steep liquor in the 15th paragraph.

17. A method for producing succinic acid, characterized in that in the 15th paragraph, the culturing is performed by adding a neutralizing agent selected from the group consisting of Ca(OH)2, KOH, NaOH, Mg(OH)2, NH4OH and a mixture of two or more thereof.

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