Indian Patents. 228001:RUMEN BACTERIA MUTANT AND A PROCESS FOR PREPARING SUCCININC ACID EMPLOYING THE SAME

Title of Invention	RUMEN BACTERIA MUTANT AND A PROCESS FOR PREPARING SUCCININC ACID EMPLOYING THE SAME
Abstract	The present invention relates to novel rumen bacterial mutants resulted from the disruption of a lactate dehydrogenase gene (ldhA) and a pyruvate formate-lyase gene (pfl) have been disrupted, and has the property of producing succinic acid at high concentration while producing little or no other organic in anaerobic conditions.

Full Text	The present invention relates to a rumen bacterial mutant which produce succinic acid at higji concentration while producing little or no other organic acids, as well as a method for producing succinic acid, which is characterized by lie culture of such mutants in anaerobic conditions. BACKGROUND ART Various anaerobic microoigamsms, including Succinivibrio dextrinosolvens, Fibrobacter succinogenes, Ruminococcus Jlavefaciens and the like, produce succinic acid as an end product by. glucose metabolism (Zeikus, Annu. Rev. Microbiol, 34:423, 1980). Strains that produce succinic acid at industrially useful yield have not yet been reported except for Anaerobiospirillum succiniciproducens known to produce succinic acid at high concentration and high yield fiom glucose upon the presence of excessive CO2 (David et al, Int. J. Syst. Bacteriol, 26:498, 1976). However, since Anaerobiospirillum succiniciproducens is an obligate anaerobic microorganism, a fermentation process of producing succinic acid using this microorganism has a shortcoming that the process itself becomes unstable even upon exposure to a very small amount of oxygen. To overcome this shortcoming, Mannheimia succiniciproducens 55E was developed that is a strain having not only resistance to oxygen but also high organic acid productivity. However, since this strain produces formic acid. acetic acid and lactic acid in addition to succinic acid, it has shortcomings in &at it has low yield and costs a great deal in a purification process of removing other organic acids except succioic acid. Recombinant E. coli strains for the production of succinic acid have been reported in various literatures. If the E. coli strains have disruptions of a gene coding for lactate dehydrogenase and a gene coding for pyruvate fonnate-lyase, it is hard for them to grow in anaerobic conditions. Furthermore, they have too low yield to apply them to industrial field, since, although lactic acid is not produced as a fermentation product, otiier metabolites (acetic acid and ethanol) account for about half of the production of succinic acid, hi an attempt to overcome such shortcomings, E. coli cells were grown in aerobic conditions, and then anaerobic conditions were applied to induce the fermentation of succinic acid. However, this attempt still has low productivity (Vemuri et al, J. Ind. Microbiol. Biotechnol, 28:325, 2002). Also, other examples were reported in which the genes of enzyrues, such as pyruvate carboxylase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and malic enzyme, that immobilize CO2 in a metabolic pathway of succinic acid fermentation, are introduced into E. coli, thereby increasmg the production of succmic acid (Vemuri et al, Appl. Enviwn. Microbiol, 68:1715, 2002; Millard et al, Appl Environ. Microbiol, 62:1808, 1996; Chao and Liao, Appl Environ. Microbiol, 59:4261, 1993; Stols andDonnelly, Appl Environ. Microbiol, 63:2695,1997). Meanwhile, it is known that the disruption of ptsG in E. coli contributes to an impmvemftTit of bacterial production and succinic acid production (Chattegee et al, Appl Environ. Microbiol, 67:148. 2001), but most of rumen bacteria have no ptsG, and thus have an advantage that they do not require a removal process of ptsG as m the case of E. coli. Recentiy, an atten^t is actively conducted in which the genes of enzymes that immobilize CO2 in a metabolic pa&way of succinic acid fermentation are introduced into rumen bacteria, including genus Actinobacillus and genus Anaerobiospirillum. However, in this attempt, other organic acids were produced at large amounts or the yield of succinic acid "Wis so low, as a result of that, it did not reach an industrially applicable level. DISCLOSURE OF INVENTION Accordingly, during onr extensive studies to develop bacterial strains that produce succinic acid at high yield, the present inventors constructed bacterial mutant Mamheimia sp. LPK (KCTC 10558BP) by the disruption of a lactate dehydrogenase gene {IdhA) and a pyruvate formate-lyase gene {pfl) from Mannheimia succiniciproducens 55E, which is a kind or rumen bacteria, and constructed bacterial mutants Mannheimia sp. LPK7 and LPK4, by the disruption of phosphotransacetylase gene iptd) and an acetate kinase gsae {adcA), and a phosphoenolpyruvate carboxylase gene (ppc\ respectively from the LPK strain, and then confirmed that the culture of such bacterial mutants in anaerobic conditions provides sucdnic acid at high yield, thereby completing ttie present invention. Therefore, a main object of the present invention is to provide a rumen bacterial mutant that produces succinic acid at high yield while producing no other organic acids, as well as a producing method thereof Another object of the present invention is to provide a method of producing sucdnic acid, which is characterized by the culture of the above bacterial mutants in anaerobic conditions. To achieve the above objects, in one aspect, the present invention provides a rumen bacterial mutant which a lactate dehydrogenase-encoding gene (IdhA) and a pyruvate fonnate-lyase-encoding gene (pfl) have been disrupted, and has the property of producing succinic acid at high concentration while producing little or no other organic acids in anaerobic conditions. In another aspect, the present invention provides a rumen bacterial mutant which a lactate dehydrogenase-encoding gene (IdkA), a pyruvate formate-lyase-encoding gene (pjl), a phosphotransacetylase-encoding gene {ptd) and a acetate kinase-encoding gene (ackA) have been disrupted, and has the property of producing succinic acid at high concentration while producing little or no other organic acids in anaerobic conditions. In still another aspect, the present invention provides a rumen bacterial mutant which a lactate dehydrogenase-encoding gene (IdkA), a pyruvate formate-lyase-encoding gene (pfl), and a phoE^ihoenolpyruvate carboxylase-encoding gene (ppc) have been disrupted, and has the property of producing succinic acid at high concentration while producing Uttle or no other oi^anic acids in anaerobic conditions. In the present invention, lie rumen bacteria are preferably homo-fermentative bacteria fliat may be selected from the group consisting of genus Mannheimia, genus Actinobacillus and genus Anaerobiospirillum and produce only succinic acid while producing Uttle or no other organic acids. In a preferred embodiment of the present invention, the rumen bacterial mutant is Mannheimia sp. LPK, LPK7orLPK4. In still another aspect, the present invention provides a method for producing rumen bacterial mutant tiiat has the property of producing succinic acid at high concentration while producing little or no other organic acids in anaerobic conditions, the method comprising disn^ting a lactate dehydrogenase-encoding gene (IdhA) and a pyruvate formate-lyase-encoding gene (pfl) from rumen bacteria that are selected from the group consisting of genus Mannheimia, genus iciinobacillus and genus Anaerobiospirilium. ■n the inventive method for producing the rumen bacterial mutant, the disruptions jf the IdhA and pfl genes are preferably performed by homologous recombination, rhe homologous recombination is preferably performed using a genetic exchange sector containing a disrupted IdkA and a genetic exchange vector containing a iisrupted pfl. Preferably, the vector containing a disrupted IdhA is pMLKO-acB, and the vector conteining a disrupted p^ is pMPKO-sacB. in yet another aspect, the present invention provides a method for producing umen bacterial mutant that has the property of producing succinic acid at high »ncentration while producing little or no other organic acids in anaerobic jonditions, the metiiod comprising additionally disrupting a )hosphotransacetylase-encoding graie iptd) and an acetate kinase-encoding gene dcM) from rumen bacteria that are selected from the group consisting of genus \iannheimia, genus Actinobacillus and genus Anaerobiospirillum, and a lactate iehydrogenase-encoding gene (JdkA) and a pyruvate foimate-lyase-encoding gene pfl) have been disrupted. ?he disruptions of the pta and ackA genes are preferably performed by lomologous recombination. The homologous recombination is preferably lerformed using a genetic exchange vector containing a disrupted/7fa and ackA. Tie genetic exchange vector containing a disrupted pta and ackA is preferably iPTA-sacB. n yet another aspect, the present invention provides a method for producing umen bacterial mutant that has &e property of producing succinic acid at high oncentration while produrang Uttle or no other organic acids in anaerobic onditions, the method comprising additionally disrupting a phosphoenolpyruvate arboxylase-encoding gaie {ppc") from rumen bacteria that are selected from the group consisting of genus Mannheimia, genus Actinobacillus and genus Anaerobiospmllum, and a lactate dehydrogenase-encoding gene {IdhA) and a pyruvate formate-lyase-encoding gene {pft) have been disrupted. The disruption of the ppc gene is preferably performed by homologous recombination. The homologous recombination is jMreferably performed using a genetic exchange vector containing a disrupted ppc. The genetic exchange vector containing a disrupted/3pc is preferably pPPC-sacB. In the present invention, the rumen bacterial mutant having disruptions of a lactate dehydrogenase-encoding gene QdhA) and a pyruvate formate-lyase-encoding gene ipfl) is preferably A/oTinAezm/a sp. LPK (KCTC 10558BP). In yet another aspect, the present invention provides a genetic exchange vector pMLKO-sacB containing a disrupted IdhA; a genetic exchange vector pMPKO-sacB containing a disrupted ^_^; a genetic exchange vector pPTA-sacB containing a disruptedpto and ackA; and a graietic exchange vector pPPC-sacB containing a disrupted/jpc. In another further aspect, the present invention provides a method for producing succinic acid, the method comprising the steps of: culturing the rumen bacterial mutants in anaerobic condition; and recovering succinic acid from the culture broth. As used herein, the tMm "disruption" means that the genes encoding the enzymes are modified such that the enzymes cannot be produced. In the present invention, each of tiie lactate dehydrogenase gene (IdhA) and the pyruvate formate-lyase gene (pfl) was identified from the genomic information of Mannheimia succiniciproducens 55E, which is a kind of rumen bacteria, and then, all the two genes were removed from Mannheimia succiniciproducens 55E using a vector having disruptions of the genes, thCTeby constructing the bacterial mutant Mannheimia sp. LPK (KCTC 10558BP). Next, each of pta-ackA genes and a ppc gene was disrupted from the bacterial mutant Mannheimia sp. LPK, thereby constructing various bacterial mutants. Then, such bacterial mutants were confirmed to produce succuiic acid at high concentration while producing little or no other organic acids. The inventive bacterial mutants (Mannheimia sp. LPK, LPK4 and LPK7) are facultative anaerobic, gram-negative, non-mobile rods or cocobacilH, do not produce endospores, and can produce succinic acid in anaerobic conditions. BRIEF DESCRIPTION OF DRAWINGS FIG. 1 shows a process of constructing a vector containing a disrupted IdhA (pMLKO-sacB). FIG. 2 shows a process of constructing a vector containing a disrupted pfl (pMPKO-sacB). FIG. 3 s\iows a process of constructing a bacterial mutant (LPK) by disrupting IdhA sadpfl gaies from Mannheimia succiniciproducens 55E. FIG. 4 is an electrophoresis photograph showing the disruption of IdhA and pfl genes from Mannheimia sp. LPK (M: lambda Hzndlll size marker, lanes 1-3: PCR product LUl & KMl (1.5 kb); lanes 4-6: PCR product LD2 & KM2 (1.7 kb); lanes 7-9; PCR product PUl & CMl (2.2 kb); and lanes 10-12: PCR product PD2 &CM2(1.6kb)). FIG. 5 shows the culture characteristics of Mannheimia sp. LPK in anaerobic conditions saturated with COj. FIG. 6 shows a process of constructing vectM: containing a disrupted p/a and ackA (pPTA-sacB). FIG. 7 is a process of constructing a vector containing a disrupted ppc (pPPC-sacB). FIG. 8 shows a process of constructmg bacterial mutant LPK7 by disrupting pta and ackA genes fix)m Mannheimia sp. LPK. FIG. 9 shows a process of constructing bacterial mutant LPK4 by disrupting a/ipc gene from Mannheimia sp. LPK. FIG. 10 is an electrophoresis photograph showing the disruption of pta and ackA genes from Mannheimia sp. LPK7 (M: 1-kb ladder size marker, lane 1: PCR product P13 & P14 (1.1 kb); and lane 2: PCR product P15 & P16 (1.5 kb)). FIG. 11 is an electrophoresis photograph showing the disruption of a ppc gene from Mannheimia sp. LPK4 (M: 1-kb ladder size marker; lane 1: PCR product P13 & P17 (1.1 kb); and lane 2: PCR product P15 & P18 (1.5 kb)). FIG. 12 shows the cultivation characteristics of Mannheimia sp. LPK7 in anaerobic conditions saturated with CO2. FIG. 13 shows the cultivation characteristics of Mannheimia sp. LPK4 in anaerobic conditions saturated with CO2. DETAILED DESCRIPTION OF THE INVENTION The present invention will hereinafter be described in fiirther detail by exan^>les. It will however be obvious to a person skilled in the art that these exan5)Ies are given for illustrative purpose only, and the present invention is not limited to or by the examples. Particularly, the following examples illustrate only a method comprising disrupting genes &om a genus Mannheimia strain to obtain bacterial mutants and then producing succinic acid at high concentration by these bacterial mutants. However, methods by which bacterial mutants having disruptions of such genes are obtained ftom other rumen bacterial strains, such as genus Actinobacillus and genus Anaerobiospirillum, and succinic acid is produced using the bacterial strains, will also be obvious to a person skilled in the art Furthermore, the following examples illustrate only a certain medium and culture method. However, the use of other mediums different from, such as whey, com steep liquor (CSL), as described in literatures (Lee et al., Bioprocess Biosyst. Eng., 26:63, 2003; Lee et al., Appl. Microbiol BiotechnoL, 58:663, 2002; Lee et al, Biotechnol Lett.^ 25:111, 2003; Lee et al, Appl Microbiol Biotechnol, 54:23, 2000; Lee et al., Biotechnol Bioeng., 72:41, 2001), and the use of various methods, such as fed-batch culture and continuous culture, will also be obvious to a person skilled in the art. Example 1: Construction of pMLKO-sacB In order to disrupt a lactate dehydrogenase gene {IdkA) by homologous recombination, a gene exchange vector was constructed in the following manner. First, the genomic DNA- of UamUieimia sucdniciproducens 55E (KCTC 0769BP), as a tenqjlate, was subjected to PCR using primers set forth in SEQ ED NO: 1 and SEQ ID NO: 2 below, and then, the obtained PCR fragmait was cut with Sacl and Pstl and introduced into pUClS (New England Biolabs, Inc., Beverly, Mass.), thereby constructing pUC 18-L1. SEP ID NO: 1-. S"-CAGTGAAGGAGCTCCGTAACGCATCCGCCG (LSI) SEP ID NO: 2: 5"-CTTTATCGAArCTGCAGGCGGTTTCCAAAA (LPl) rhereafter, the genomic DNA of Mannheimia succiniciproducens 55E, as a emplate, was subjected to PCR using primers set forth in SEQ ID NO: 3 and SEQ DD NO: 4 below, and the resulting PCR fragment was cut with Pstl and HindSl md introduced into the pUC 18-L I, thereby constructing pUCl 8-L 1-L2. SEQ ID NO: 3: S"-GTACTGTAAACTGCAGCTTTCATAGnAGC (LP2) SEQ ID NO: 4: 5"-GCCGAAAGTCAAGCTTGCCGTCGTTTAGTG (LH2) )UC4K (Pharmacia, Freibui^, Germany) was cut with Pstl, and the resulting anamycin-resistant gene was fiised with pUC18-Ll-L2 cut with Pstl, thereby instructing pUC18-Ll-KmR-L2. A linker set forth in SEQ ID NO; 5 was Dserted into the pUC18-Ll-KniR-L2 cut with Sacl, thereby making a new A&al utting site. SEP ID NO: 5: 5"-TCTAGAAGCT "CR on pKmobsacB (Schafer et al. Gene, 145:69, 1994) as a template was crformed using primers set forth in SEQ ID NO: 6 and 7 below, and the resulting "CR product was cut with JCbal and inserted into the above Xbal restriction nzyme site, thereby constructing pMLKO-sacB (FIG. 1). SEP ID NO: 6: S"-GCTCTAGACCTTCTATCGCCTTCTTGACG (SXF) SEP ID NO: 7: S"-GCTCTAGAGGCTACAAAATCACGGGCGTC (SXR) Ixample 2: Construction of pMPKO-sacB 1 ordei to disrupt a pyruvate formate-lyase gene (pjt) by homologous ■ecombination, a genetic exchange vector was constructed in the following nanner. A pKmobsacB template containing a sacB gene (Genbank 02730) was jubjected to PCR using primers set forth in SEQ ID NO: 8 and SEQ ID NO: 9 >elow. The resulting sacB product was cut with Pstl and BamSi and inserted nto pUC19 (Stratagene Cloning Systems. La JoUa, Calif.), ihsrehy constructing )UC19-sacB. SEP ro NO: 8: 5"-AGCGGArCCCCTTCTATCGCCTTCTTGACG (SBG) SEP ID NO: 9: 5"-GTCCTGCAGGGCTACAAAATCACGGGCGTC (SPR) rhe genomic DNA of Mannheimia succiniciproducens 55E, as a template, was rubjected to PCR using primers set forth in SEQ ID NO: 10 and SEQ ID NO: 11 >elow. The resulting PCR fragment was cut witii BamYQ. and fused with the 3UC19-sacB cut with BamW.^ thereby constructing pUC19-sacB-pfl. SEC ID NO: 10: 5"-CATGGCGGATCCAGGTACGCTGAnTCGAr (PBl) SEOIDNQ:!!: S"-CAAGGATCCAACGGATAAAGCmTAITATCPB2) n order to obtain a chloramphenicol-resistant gene, pACYCI84 (New England 3iolabs, Inc., Beverly, Mass.) as a ternplate was subjected to PCR using primers jet forth in SEQ ID NO: 12 and SEQ ID NO: 13 below. The resulting PCR DToduct was cut with Smdl and fused with the pUC19-sacB-pfl cut with BstWQTl, hereby constmcting pMPKO-sacB (FIG 2). SEP ID NO: 12: SVCTCGAGCCCGGGGTTTAAGGGCACCAAIAA (CTR) SEP ID NO: 13: S"-CTCGAGCCCCGGGCnTGCGCCGAATAAAT (GIF) Example 3: Construction oj Mannheimia sp. LPK strain ?IG 3 shows a process of constructing a mutant strain (LPK) by disrupting IdhA md pfl genes from Mannheimia succiniciproducens 55E. Mannheimia "succiniciproducens 55E was plated on LB-glucose medium containing 10 g/1 of glucose, and cultured at 37°C for 36 hours. The colony formed was inoculated in 10 ml of LB-glucose liquid medium, and cultured for 12 hours. The culture broth which had been sufficiently grown was inoculated by 1% in 100 ml of LB-ghicose liquid medium, and cultured in a shaking incubator at 200 ipm and "iT"C. When the culture broth reached an OD of about 0.2-0.3 after 4-5hours, it was centriiuged at 40 and 4000 ipm for 10 minutes to collect cells. Then, the cells were resuspended in 200 nil of 10% glycerol solution at 4°C. The suspension was centriiuged at 4"C and 4000 ipm for 10 minutes, and tiie cells were collected and resuspended in 200 ml of 10% glycerol solution at 4""C, and then centriiuged at 4°C and 4000ipm for 10 miimtes to collect the cells. The cells were suspended in glycerol at a volume ratio of 1:1, to obtain cell concentrate. The cell concentrate thus obtained was mixed with the genetic exchange vectors pMLKO-sacB and pMPKO-sacB constructed in Stamples I and 2, and then subjected to electroporation under conditions of 1.8 kV, 25 ^F and 200 ohms. 1 ml of LB-glucose hquid medium was added to the electroporated mixture and cultured in a shaking incubator at 37°C and lOOrpm for one hour. The culture broth was plated on LB-glucose soHd medium contaming a suitable antibiotic [Km (final concaitration of 25 lagtail) or Cm (6.8 jig/ml) and cultured at 37°C for 48 hours or more. In order to select a colony ^here only double crossover occurred, ths colonies formed were streaked on LB-sucrose medium (LB medium with IOOg/1 suCTOse) containing Km 25 Hg/ml) or Cm (6.8\|ag/ml). Ailer 24 hours, the formed colonies were streaked again on the same plate. The colony (mutant) formed on the plate were cultured in LB-glucose liquid medium containing an antibiotic, and a genomic DNA was isolated firom the cultured strain by the method described in Rochelle et al. (FEMS Microbiol. Lett., 100:59, 1992). PCR was performed using the isolated mutant genomic DNA as a template, and the PCR product was electrophoresed to confirm the disruption of IdhA and/?/? genes from the PCR product hi order to confirm the disruption of tiie IdkA gene, PCRs were performed twice m the following manners. First, the mutant genomic DNA as a teai^late was subjected to PCR using primers set forth in SEQ ID NO: 14 and SEQ ID NO: 15. SEC ID NO: 14: S"-GACGnTCCCGTTGAATATGGC (KMl) SEC ID NO: 15: S"-CArTGAGGCGTATTATCAGGAAAC (LUl) Then, the mutant genomic DNA as a template was subjected to PCR using primers set forth in SEQ ID NO: 16 and SEQ ID NO: 17 below. The products obtained m flie two PCRs were subjected to gel electrophoresis to confirm the disruption of IdhA by their size (1.5 kb) (FIG 4). SEP ID NO: 16: 5"-GCAGTTTCArTTGArGCTCGArG (KM2) SEP ID NO: 17: 5"-CCTCTrACGArGACGCATCTTTCC (LD2) In order to confirm the disruption of pfl, PCRs were performed twice in the following manner. First, the mutant genomic DNA as a template was subjected to PCR using primers set forth in SEQ ID NO: 18 and SEQ ID NO: 19 below. SEP ID NO: 18: S"-GGTGGTATATCCAGTGATTTmTCTCCAT (CMl) SEP ID NO: 19:5"-CTTTGCAACATTATGGTATGTATTGCCG (PUI) Then, the mutant genomic DNA as a template was subjected to PCR using primeis set forth in SEQ ID NO: 20 and SEQ ID NO: 21. The products obtained in tiie two PCRs were subjected to gel electrophoresis to confirm the disruption of pfl by their size (l.Skb) (FIG. 4). In FIG. 4, M represents a Lambda Htndlll size maricer, lanes 1-3 represent Ihe PCR product LUl & KMl (1.5kb), lanes 4-6 represent the PCR product LD2 & KM2 (l.Tkb), lanes 7-9 represent the PCR product PUI & CM.I (2.2kb), and lanes 10-12 represent the PCR product PD2 & CM2 (1.6kb). SEOIDNO:20:5"-TACTGCGATGAGTGGCAGGGCGGGGCGTAA (CM2) SEP ID NO: 21:5"-CCCCAGCATGTGCAAATCTTCGTCAC (PD2) The disruption of IdhA was confirmed by the fact that the product resulted fix)m the PCR using the primers (LUl and KMl) of SEQ ID NO: 14 and SEQ ED NO: 15 has a size of 1.5 kb an at tiie same time the product resulted from the PCR using the primers (LD2 and KM2) of SEQ ID NO; 16 and SEQ ID NO: 17 has a size of 1.7 kb. And, the disnqjtion of pfl was confirmed by the fact that the product resulted fixim the PCR using the primers (PUl and CM!) of SEQ ID NO; 18 and SEQ ID NO: 19 has a size of 22 kb and at die same time the product resulted from the PCR using the primers (PD2 and CM2) of SEQ ID NO; 20 and SEQ ID NO: 21 has a size of 1.6 kb. The position of each primer is shown in FIG. 3. The mutant constructed by the above method, i.e., a bacterial mutant having disruptions of IdhA and pjl, was named "Mannheimia sp. LPK" and deposited under accession number KCTC 1088 IBP on November 26, 2003 in the Korean Collection for Type Cultures (KCTC), Korean Research Institute of Bioscience and Biotechnology (KRIBB). Example 4: Fermentation characteristics of Mannheimia sp. LPK In order to examine the fermentation charactaistics of Mannheimia sp. LPK constructed in Example 3 above, the mutant was cultured in anaerobic conditions saturated with CO2, and the resulting reaction product was analyzed. First, caibon dioxide was introduced into 100 ml of preculture medium consisting of 20g/L glucose, 5g/L polypeptone, 5g/L yeast extract, 3g/L K2HPO4, Ig/L NaCl, Ig/L (NH4)2S04, 0.2g/L CaClz • 2H2O, 0.2g/L MgClz • 6H2O and lOg/L MgCOa, and then, Mannheimia sp. LPK was inoculated in ttie preculture medium and precultured at 39C for 14 hours. Then, 0.9 L of culture medium consisting of 20g/L glucose, 5g/L polypeptone, 5g/L yeast extract, 3g/L K2HPO4, Ig/L NaQ, 5g/L (NH4)2S04, 0.2g/L CaQj - 2H2O, 0.2g/L MgClj • 6H2O and 5g/L NajCOa was put in a 2.5-L culture tanV^ and 100 ml of the precultured microorganisms were inoculated in the culture medium and batch-cultured at SP^C and pH 6.5 while supplying carbon dioxide at a flow rate of 0.25vvm. The concentration of cells in the culture broth was measured with a spectrophotometer (Ultraspec 3000, Pharmacia Biotech., Sweden), and the amounts of succinate, glucose, lactate, acetate and fonnate were measured by HPLC (Aminex HPX-87H column, Bio-Rad, USA) (FIG. 5). Symbols in FIG- 5, refer to changes in the concentrations of cells (•), succinate (o), glucose (■), formate (O) and acetate (A) with the passage of culture time. As shown in FIG. 5, after 14 hours of culture, the concentration of consumed glucose was 20g/L and the concentration of produced succinate was 17.2g/L, indicating that the yield of succinate (the amount of produced succinate/the amount of consumed glucose) is 31% and the volume productivity of succinate (the concentration of produced niccinate/elapsed time) is 1.23 g/L/h. The inventive mefliod of prwlucing juccinic acid by culturing Mannheimia sp. LPK in anaerobic conditions saturated vith CO2 showed a great increase in yield as compared to tiiat of producing tuccinic acid by culturing parent strain Mannheimia succiniciproducens 55E in maCTobic conditions saturated with CO2, and showed a ratio of succinic acid : icetic acid of 40.7:1, indicating that it can produce succinic acid mth little or no )y-IHX)ducts. iliample 5: Construction of pPTA-sacB n order to disrupt a pho^hotransacetylase gene (ptd) and an acetate kinase gene ackA) by homologous recombinatioiL, a genetic exchange vector was constructed Q the following manner- First, the genomic DNA of Mannheimia sp. LPK KCTC 10558BP), as a template, was amplified by PCR using primers set forth in ;EQ ID NO: 22 and SEQ K) NO: 23 below, and the resulting PCR fiagment was ut with Xbal and 5amHI and introduced into pUC19, thereby constructing 1UCI9-PTAI. SEP ID NO: 22: 5"-GCTCTAGATATCCGCAGTATCACTTTCTGCGC SEP ID NO: 23: S"-TCCGCAGTCGGATCCGGGTTAACCGCACAG Thereafter, the genomic DNA of Mannheimia sp. LPK as a template was amplified by PCR using primers set forth in SEQ ID NO: 24 and SEQ ID NO: 25 below, and the resulting PCR fragment was cut with Xbal and Sad and introduced into the pUC19-PTAl, thereby constructing pUC19-PTAl2. SEP ID NP: 24: 5"-GGGGAGCTCGCTAACITAGCrTCTAAAGGCCATGT TTCC SEP ID NO: 25: 5"-GCTCTAGATATCCGGGTCAATATCGCCGCAAC As a template, plasmid pIC156 (Steimnetz et al. Gene, 142:79, 1994) containing a spcctinomycin-resistant gene (GenBank X02588) was amplified by PCR using primers set forth in SEQ ID NO: 26 and SEQ ID NO: 27 below, and the resulting PCR firagment (spectinomycin-resistant gene) was cut with EcdBN and introduced into the pUC19-PTA12, thereby constructing pUC19-PTAlS2 having the spectinomycin-resistant gene. The constructed pUC19-PTAlS2 was cut with JSQCI and BamHI and introduced into pUC19-SacB (see Example 2), thereby constructing a pPTA-sacB vector (FIG. 6). SEP ID NP: 26: 5"-GAATTCGAGCTCGCCCGGGGATCGATCCTC SEP ID NP: 27: 5"-CCCGGGCCGACAGGCTTTGAAGCATGCAAATGTCAC Example 6: Construction of pPPC-simB [n order to disrupt a phosphoenolpyruvate carboxylase gene {ppc) by homologous recombination, a genetic exchange vector was constructed in the following [naimer. First, the genomic DNA of Mannheimia sp. LPK, as a template, was unplified by PCR using primers set forth in SEQ ID NO: 28 and SEQ ID NO: 29, md die resulting PCR fragment was cut with Xbal and BamSi and introduced nto pUC19, thereby constructing pUC19-PPCl. SEP ID NO: 28: S"-TACGGATCCCCAGAAAATCGCCCCCATflTCGA SEP ID NO: 29-. S"-GCTCTAGATATCGTTTGATATTGTTCCGCCACATrTG Thereafter, the genomic DNA of Mannheimia sp. LPK, as a template, was subjected to PCR using primers set forth in SEQ ID NO: 30 and SEQ ID NO: 31, and the resulting PCR fragment was cut with JOyal and Sad and introduced into the pUC19-PPCl, thereby constractmg pUC19-PPC12. SEO ID NO: 30:5"-GCTCTAGATATCCGTCAGGAAAGCACCCGCCATAGC SEC ID NO: 31: 5-GGG A spectinomycin-resistant gene cut with EcoVN (see Example 5) was introduced into the pUC19-PPC12 to conshiict pUC19-PPClS2. The pUC19-PPClS2 was cut with Sad and BamSl and introduced into the pUC19-SacB, thereby constructing a pPPC-sacB vector (FIG. 7). Example 7: Construction of Mannheimia sp. LPK7 and LPK4 strains . FIG. 8 and FIG. 9 show processes of constructing mutant strains LPK7 and LPK4 by disrupting pta-ackA. and ppc fii>m Mannheimia sp. LPK, respectively. Mannheimia sp. LPK was plated on LB-glucose medium containing lOgA glucose, and cultured at 3>TC for 36 hours. The colony formed was inoculated in 10 ml LB-glucose hquid medium and cultured for 12 hours. The culture broth which had been sufficiently grown was inoculated by 1% in 100 ml LB-glucose liquid medium and cultured in a shaking incubator at 37"C. Cell concentrate was collected from the resulting culture broth in the same manner as descaibed in Example 3. The collected cell concentrate was mixed with the genetic exchange vectors pPTA-sacB and pPPC-sacB constructed in Exanqiles 5 and 6, and then subjected to electroporation under conditions of 1.8 kV, 75°? and 200 ohms. The electroporated mixture was added with 1 ml of LB-glucose liquid medium and cultured in a shaking incubator at 200 rpm and 37"C for one hour. The culture broth was plated on LB-glucose solid mediimi containing a spectinomycin antibiotic (final concentration: 50 (g/ml), and cultured at 37°C for at least 48 hoiurs. In order to select a colony where double crossover occurred, the colonies formed were streaked on LB-sucrose medium (LB mediiun containing 100 g/1 of sucrose) containing 50 (g/ml of spectinomycin. After 24 hours, the formed colonies were re-streaked on the same plate. The colony (mutant) formed on the plate was cultured in LB-glucose hquid medium containing an antibiotic, and a genomic DNA was isolated fiT>m the cultured strain by the method of Rochelle et al. The isolated mutant genomic DNA as a template was anqjlified by PCR, and the PCR product was electrophoresed to confirm the disruption of each of pta-ackA andppc. To confirm flie disruption of pta-ackA, PCRs were performed twice in the following manner. First, the mutant genomic DNA as a template was subjected to PCR using primers set forfii in SEQ ID NO: 32 and SEQ ID NO: 33 below. Then, the mutant genomic DNA as a ten^late was subjected to PCR using primers set forth in SEQ ID NO: 34 and SEQ ID NO: 35. SEC ID NO: 32: 5"-CCTGCAGGCATGCAAGCTTGGGCTGCAGGTCGACTC SEP ID NO: 33: S"-GCTGCCAAACAACCGAAAATACCGCAATAAACGGC SEQ ID NO: 34: S"-GGATGTAACnTACTGGATATAGCTAGAAAAGaCATCGaGGAG SEP ID NO: 35: S"-GCAACGCGAGGGTCAATACCGAAGGATTTCGCCG The products obtained in the two PCRs WCTC subjected to gel electrophoresis to confirm ttie disruption of pta-ackA by their size (FIG. 10). In FIG. 10, M represents a 1-kb ladder size marker, lane 1 rejiresents the PCR product P13 & P14 (1.1 kb), and lane 2 represents the PCR product P15 & P16 (1.5 kb). The disruption of pta-ackA was confirmed fay the fact the product resulted fix>m the PCR using the primers of SEQ ID NO: 32 and SEQ ID NO: 33 (P13 & P14) has a size of 1.1 kb at flie same time the product resulted from the PCR using the primers of SEQ ID NO: 34 and SEQ ID NO: 35 (P15 & P16) has a size of 1.5 kb. The positions of the primers are shown in FIG. 8. The mutant strain constructed as described above, i.e., a strain resulted from tiie disruption of pta-ackA from Mannheimia sp. LPK, was named "Mannheimia sp. LPK7" and deposited under accession number "KCTC 10626BP" in KCTC, an international depositary authority. Furthermore, to confirm the disruption of ppc, PCRs were performed twice in the following msama. First, the mutant genomic DNA as a template was subjected to PCR using primers set forth in SEQ ID NO: 32 and SEQ ID NPO: 36. Then, the mutant genomic DNA as a template was subjected to PCR using primers set forth in SEQ ID NO: 34 and SEQ ID NO: 37. SEC IP NO: 36: 5"-GATCCAGGGAATGGCACGCAGGCTTrCAACGCCGCC SEC ID NO: 37: S"-GCAAAGCCAGAGGAATGGATGCCATTAACCAATAGCG The products obtained in the two PCRs were subjected to gel electrophoresis to confirm (he disruption of ppc by flieir size (FIG. 11). In FIG. 11, M represents a 1-kb ladder size marker, lane 1 is the PCR product P13 & P17 (l.lkb), and lane 2 represents the PCR product PIS & P18 (1.5kb). The disruption of ppc was confirmed by the fact that the product resulted from the PCR using the primers of SEQ ID NO: 32 and SEQ ID NO: 36 (P13 & PIT) has a size of 1.1 kb at the same time the product resulted from the PCR using the primflers of SEQ ID NO: 34 and SEQ ID NO: 37 (P15 & P18) has a size of 1.5 kb. The positions of the primers are shown in FIG. 9. The mutant sti^iin constructed as descdbed above, i.e., a strain resulted from the disruption of ppc from Mannheimia sp. LPK, was named "Mannheimia sp. LPK4". Example 8: Fermentation characteristics of LPK7 and LPK4 hi order to examine the fermentation characteristics of Mannheimia sp. LPK7 and LPK4 constructed in Example 7 above, the mutant strains were cultured m anaerobic conditions saturated with CO2, and the resulting reaction products were aoalyzed. First, carbon dioxide was introduced into 200inl of the preculture mediimi as described in Example 4, and each of Mannheimia sp. LPK7 and LPK4 was inoculated in the precultuxe medium and precultured at 39*C for 24 hours. Next, 1.8 L of a culture medium, which is the same as that in Exan^le 4 except that glucose concentration is 18 g/L (final lOOniNf), was put in a 6.6 L culture tank, and 100 ml of the precultured microorganisms was inoculated in the culture medium and then batch-cultured at 39°C and pH 6.5 while supplying carbon dioxide at a flow rate of 0.25wm. The concentrations of cells, succinate, glucose, lactate, acetate and formate were measured ia the same mamier as in Example 4 (FIG. 12 and FIG. 13). Symbols in FIG. 12 and FIG. 13 refer to changes in the concMitrations of cells (• m upper portion), succinate (• m lower porticm), glucose (a), formate (♦) and acetate (A) with the passage of culture time. As shown in FIG. 12, after 22 hours of the culture of Mannheimia sp. LPK7, the concentration of consumed glucose was lOOmM and the concentration of produced succinate was 124mM, indicating that the yield of succinate (the amount of produced succinate/the amount of consumed glucose) is 124 mol%. And, the production of acetate was remarkably reduced (Table 1). Also, as shown in FIG. 13, after 22 hours of the culture of Mannheimia sp. LPK4, the concentration of consumed glucose was lOOmM and the concentration of produced succinate was 123.7mM, indicating that the yield of succinate (the amount of produced succinate/the amount of consumed glucose) is 123.7 mol%. And, the production of acetate was greatly reduced as conqsared to tiiat m the wild type (Table 1). iVhile &e present invention has been described in detail with Teforence to the T>ecific features, it will be ^jparent to persons skilled in the art tiiat fiiis iescription is only for a preferred embodiment and does not limit the scope of the )resent invention. Thus, the substantial scope of the present invention will be iefined by the appended claims and equivalents thereof. INDUSTRIAL APPLICABILITY As described and provided above in detail, Mannheimia sp. mutant strains (LPK, LPK7 and LPK4) produce succinic acid in anaerobic conditions saturated with CO2 and are facultative anaerobic strains having high resistance to oxygen. Thus, tiie production of succinic acid using such mutants can not only eliminate the fCTmaitation process instability caused by oxygen exposure, etc., but also eliminate the production of other organic acids, as con^pared to the prior method of producing succinic acid using obligate anaerobic strains, thereby making it possible to cq)timize and maximize a purification process and production yield. WE CLAIM : 1. A rumen bacterial mutant which a lactate dehydrogenase-encoding gene (IdhA) and a pyruvate formate-lyase-encoding gene (p_/I) have been disrupted, and has the property of producing succinic acid in anaerobic conditions, wherein the rumen bacteria are selected from the group consisting of genus Mannheimia, genus Actinobacillus and genus Anaerobiospirillum. 2. The rumen bacterial mutant as claimed in claim 1, wherein a phosphotransacetylase-encoding gene (pla) and an acetate kinase-encoding gene (ackA) have additionally been disrupted from the said rumen bacteria that a lactate dehydrogenase-encoding gene (IdhA) and a pyruvate formate-lyase-encoding gene (pJT) have been disrupted. 3. The rumen bacterial mutant as claimed in claim I, wherein a phosphoenolpyruvate carboxylase-encoding gene (ppc) has additionally been disrupted from the said rumen bacteria that a lactate dehydrogenase-encoding gene {IdhA) and a pyruvate formate-lyase-encoding gene (pJT) have been disrupted. 4. The rumen bacterial mutant as claimed in any one of claims 1 to 3, wherein the rumen bacteria are homo-fermentative bacteria that produce only succinic acid while producing little or no other organic acids. 5. The rumen bacterial mutant as claimed in claim 1, wherein the rumen bacterial mutant is Mannheimia sp. LPK. 6. The rumen bacterial mutant as claimed in claim 5, wherein said Mannheimia sp. LPK is KCTC 10558BP. 7. The rumen bacterial mutant as claimed in claim 2, wherein the rumen bacterial mutant is Mannheimia sp. LPK7. 8. The rumen bacterial mutant as claimed in claim 7, wherein said Mannheimia sp. LPK7 is KCTC 10626BP. 9. The rumen bacterial mutant as claimed in claim 3, wherein the rumen bacterial mutant is Mannheimia sp. LPK4. 10. A method for producing rumen bacterial mutant that has the property of producing succinic acid in anaerobic conditions, the method comprising disrupting a lactate dehydrogenase-encoding gene (JdhA) and a pyruvate formate-lyase-encoding gene {pfl) fitjm rumen bacteria that are selected from the group consisting of genus Mannheimia, genus Actinobacillus and genus Anaerobiospirillum. 11. The method for producing rumen bacterial mutant as claimed in claim 10, wherein the method comprising additionally disrupting a phosphotransacetylase-encoding gene (pla) and an acetate kinase-encoding gene (ackA) from the said rumen bacteria that a lactate dehydrogenase-encoding gene (JdhA) and a pyruvate formate-lyase-encoding gene (pjl) have been disrupted. 12. The method for producing rumen bacterial mutant as claimed in claim 10, wherein the method comprising additionally disrupting a phosphoenolpyruvate carboxylase-encoding gene (ppc) from the said rumen bacteria that a lactate dehydrogenase-encoding gene (IdhA) and a pyruvate formate-lyase-encoding gene (p/l) have been disrupted. 13. The method for producing the rumen bacterial mutant as claimed in claim 11 or 12, wherein the rumen bacterial mutant having disruptions of a lactate dehydrogenase-encoding gene (IdhA) and a pyruvate formate-lyase-encoding gene (pfl) is Mannheimia sp. LPK (KCTC 10558BP). 14. The method for producing the rumen bacterial mutant as claimed in claim 10, wherein the disruption of the IdhA and pfJ genes is performed by homologous recombination. 15. The method for producing the rumen bacterial mutant as claimed in claim 14, wherein the homologous recombination is perfonned using a genetic exchange vector containing a disrupted idkA and a genetic exchange vector containing a disrupted/?//. 16. The method for producing the rumen bacterial mutant as claimed in claim 15, wherein the genetic exchange vector containing a disrupted IdhA is pMLKO-sacB, and the genetic exchange vector containing a disrupted p/l is pMPKO-sacB. 17. The method for producii^ the rumen bacterial mutant as claimed in claim II, wherein the disruption oflhepla and ackA genes is performed by homologous recombination. 18. The method for producing the rumen bacterial mutant as claimed in claim 17, wherein the homologous recombination is perfonned using a genetic exchange vector containing a disruptedpfa and ackA. 19. The method for producing the rumen bacterial mutant as claimed in claim 18, wherem the genetic exchange vector containing a disrupted pta and ackA is pPTA-sacB. 20. The method for producing the rumen bacterial mutant as claimed in claim 12, wherein the disruption of the ppc gene is performed by homologous recombination. 21. The method for producing the rumen bacterial mutant as claimed in claim 20, wherein the homologous recombination is performed using a genetic exchange vector contaming a disrupted/Tpc. 22. The method for producing the rumen bacterial mutant as claimed in claim 21, wherein the genetic exchange vector containing a disrupted/?/)c is pPPC-sacB. 23. A genetic exchange vector pMLKO-sacB containing a disrupted IdhA. 24. A genetic exchange vector pMPKO-sacB containing a disrupted/?//. 25. A genetic exchange pPTA-sacB containing a disruptedpto and ackA. 26. A genetic exchange vector pPPC-sacB containing a disrupted ppc. 27. A method for producing succinic acid, the method comprising the steps of: culturing the rumen bacterial mutant as claimed in any one claim among claims 1 to 3 in anaerobic condition; and recovering succinic acid from the culture broth. 28. The method for producing succinic acid as claimed in claim 27, wherein the culturing step is homo-fermentation which produce succinic acid. 29. The method for producing succinic acid according to claim 27, wherein the rumen bacterial mutant is Mannheimia sp. LPK, LPK7 or LPK 4.

Full Text

The present invention relates to a rumen bacterial mutant which produce succinic acid at higji concentration while producing little or no other organic acids, as well as a method for producing succinic acid, which is characterized by lie culture of such mutants in anaerobic conditions.
BACKGROUND ART
Various anaerobic microoigamsms, including Succinivibrio dextrinosolvens, Fibrobacter succinogenes, Ruminococcus Jlavefaciens and the like, produce succinic acid as an end product by. glucose metabolism (Zeikus, Annu. Rev. Microbiol, 34:423, 1980). Strains that produce succinic acid at industrially useful yield have not yet been reported except for Anaerobiospirillum succiniciproducens known to produce succinic acid at high concentration and high yield fiom glucose upon the presence of excessive CO2 (David et al, Int. J. Syst. Bacteriol, 26:498, 1976). However, since Anaerobiospirillum succiniciproducens is an obligate anaerobic microorganism, a fermentation process of producing succinic acid using this microorganism has a shortcoming that the process itself becomes unstable even upon exposure to a very small amount of oxygen.
To overcome this shortcoming, Mannheimia succiniciproducens 55E was developed that is a strain having not only resistance to oxygen but also high organic acid productivity. However, since this strain produces formic acid.

acetic acid and lactic acid in addition to succinic acid, it has shortcomings in &at it has low yield and costs a great deal in a purification process of removing other organic acids except succioic acid.
Recombinant E. coli strains for the production of succinic acid have been reported in various literatures. If the E. coli strains have disruptions of a gene coding for lactate dehydrogenase and a gene coding for pyruvate fonnate-lyase, it is hard for them to grow in anaerobic conditions. Furthermore, they have too low yield to apply them to industrial field, since, although lactic acid is not produced as a fermentation product, otiier metabolites (acetic acid and ethanol) account for about half of the production of succinic acid, hi an attempt to overcome such shortcomings, E. coli cells were grown in aerobic conditions, and then anaerobic conditions were applied to induce the fermentation of succinic acid. However, this attempt still has low productivity (Vemuri et al, J. Ind. Microbiol. Biotechnol, 28:325, 2002). Also, other examples were reported in which the genes of enzyrues, such as pyruvate carboxylase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and malic enzyme, that immobilize CO2 in a metabolic pathway of succinic acid fermentation, are introduced into E. coli, thereby increasmg the production of succmic acid (Vemuri et al, Appl. Enviwn. Microbiol, 68:1715, 2002; Millard et al, Appl Environ. Microbiol, 62:1808, 1996; Chao and Liao, Appl Environ. Microbiol, 59:4261, 1993; Stols andDonnelly, Appl Environ. Microbiol, 63:2695,1997).
Meanwhile, it is known that the disruption of ptsG in E. coli contributes to an impmvemftTit of bacterial production and succinic acid production (Chattegee et al, Appl Environ. Microbiol, 67:148. 2001), but most of rumen bacteria have no ptsG, and thus have an advantage that they do not require a removal process of ptsG as m the case of E. coli. Recentiy, an atten^t is actively conducted in which the genes of enzymes that immobilize CO2 in a metabolic pa&way of succinic acid fermentation are introduced into rumen bacteria, including genus

Actinobacillus and genus Anaerobiospirillum. However, in this attempt, other organic acids were produced at large amounts or the yield of succinic acid "Wis so low, as a result of that, it did not reach an industrially applicable level.
DISCLOSURE OF INVENTION
Accordingly, during onr extensive studies to develop bacterial strains that produce succinic acid at high yield, the present inventors constructed bacterial mutant Mamheimia sp. LPK (KCTC 10558BP) by the disruption of a lactate dehydrogenase gene {IdhA) and a pyruvate formate-lyase gene {pfl) from Mannheimia succiniciproducens 55E, which is a kind or rumen bacteria, and constructed bacterial mutants Mannheimia sp. LPK7 and LPK4, by the disruption of phosphotransacetylase gene iptd) and an acetate kinase gsae {adcA), and a phosphoenolpyruvate carboxylase gene (ppc\ respectively from the LPK strain, and then confirmed that the culture of such bacterial mutants in anaerobic conditions provides sucdnic acid at high yield, thereby completing ttie present invention.
Therefore, a main object of the present invention is to provide a rumen bacterial mutant that produces succinic acid at high yield while producing no other organic acids, as well as a producing method thereof
Another object of the present invention is to provide a method of producing sucdnic acid, which is characterized by the culture of the above bacterial mutants in anaerobic conditions.
To achieve the above objects, in one aspect, the present invention provides a rumen bacterial mutant which a lactate dehydrogenase-encoding gene (IdhA) and a pyruvate fonnate-lyase-encoding gene (pfl) have been disrupted, and has the

property of producing succinic acid at high concentration while producing little or no other organic acids in anaerobic conditions.
In another aspect, the present invention provides a rumen bacterial mutant which a lactate dehydrogenase-encoding gene (IdkA), a pyruvate formate-lyase-encoding gene (pjl), a phosphotransacetylase-encoding gene {ptd) and a acetate kinase-encoding gene (ackA) have been disrupted, and has the property of producing succinic acid at high concentration while producing little or no other organic acids in anaerobic conditions.
In still another aspect, the present invention provides a rumen bacterial mutant which a lactate dehydrogenase-encoding gene (IdkA), a pyruvate formate-lyase-encoding gene (pfl), and a phoE^ihoenolpyruvate carboxylase-encoding gene (ppc) have been disrupted, and has the property of producing succinic acid at high concentration while producing Uttle or no other oi^anic acids in anaerobic conditions.
In the present invention, lie rumen bacteria are preferably homo-fermentative bacteria fliat may be selected from the group consisting of genus Mannheimia, genus Actinobacillus and genus Anaerobiospirillum and produce only succinic acid while producing Uttle or no other organic acids. In a preferred embodiment of the present invention, the rumen bacterial mutant is Mannheimia sp. LPK, LPK7orLPK4.
In still another aspect, the present invention provides a method for producing rumen bacterial mutant tiiat has the property of producing succinic acid at high concentration while producing little or no other organic acids in anaerobic conditions, the method comprising disn^ting a lactate dehydrogenase-encoding gene (IdhA) and a pyruvate formate-lyase-encoding gene (pfl) from rumen bacteria that are selected from the group consisting of genus Mannheimia, genus

iciinobacillus and genus Anaerobiospirilium.
■n the inventive method for producing the rumen bacterial mutant, the disruptions jf the IdhA and pfl genes are preferably performed by homologous recombination, rhe homologous recombination is preferably performed using a genetic exchange sector containing a disrupted IdkA and a genetic exchange vector containing a iisrupted pfl. Preferably, the vector containing a disrupted IdhA is pMLKO-acB, and the vector conteining a disrupted p^ is pMPKO-sacB.
in yet another aspect, the present invention provides a method for producing umen bacterial mutant that has the property of producing succinic acid at high »ncentration while producing little or no other organic acids in anaerobic jonditions, the metiiod comprising additionally disrupting a )hosphotransacetylase-encoding graie iptd) and an acetate kinase-encoding gene dcM) from rumen bacteria that are selected from the group consisting of genus \iannheimia, genus Actinobacillus and genus Anaerobiospirillum, and a lactate iehydrogenase-encoding gene (JdkA) and a pyruvate foimate-lyase-encoding gene pfl) have been disrupted.
?he disruptions of the pta and ackA genes are preferably performed by lomologous recombination. The homologous recombination is preferably lerformed using a genetic exchange vector containing a disrupted/7fa and ackA. Tie genetic exchange vector containing a disrupted pta and ackA is preferably iPTA-sacB.
n yet another aspect, the present invention provides a method for producing umen bacterial mutant that has &e property of producing succinic acid at high oncentration while produrang Uttle or no other organic acids in anaerobic onditions, the method comprising additionally disrupting a phosphoenolpyruvate arboxylase-encoding gaie {ppc") from rumen bacteria that are selected from the

group consisting of genus Mannheimia, genus Actinobacillus and genus Anaerobiospmllum, and a lactate dehydrogenase-encoding gene {IdhA) and a pyruvate formate-lyase-encoding gene {pft) have been disrupted.
The disruption of the ppc gene is preferably performed by homologous recombination. The homologous recombination is jMreferably performed using a genetic exchange vector containing a disrupted ppc. The genetic exchange vector containing a disrupted/3pc is preferably pPPC-sacB.
In the present invention, the rumen bacterial mutant having disruptions of a lactate dehydrogenase-encoding gene QdhA) and a pyruvate formate-lyase-encoding gene ipfl) is preferably A/oTinAezm/a sp. LPK (KCTC 10558BP).
In yet another aspect, the present invention provides a genetic exchange vector pMLKO-sacB containing a disrupted IdhA; a genetic exchange vector pMPKO-sacB containing a disrupted ^_^; a genetic exchange vector pPTA-sacB containing a disruptedpto and ackA; and a graietic exchange vector pPPC-sacB containing a disrupted/jpc.
In another further aspect, the present invention provides a method for producing succinic acid, the method comprising the steps of: culturing the rumen bacterial mutants in anaerobic condition; and recovering succinic acid from the culture broth.
As used herein, the tMm "disruption" means that the genes encoding the enzymes are modified such that the enzymes cannot be produced.
In the present invention, each of tiie lactate dehydrogenase gene (IdhA) and the pyruvate formate-lyase gene (pfl) was identified from the genomic information of Mannheimia succiniciproducens 55E, which is a kind of rumen bacteria, and then,

all the two genes were removed from Mannheimia succiniciproducens 55E using a vector having disruptions of the genes, thCTeby constructing the bacterial mutant Mannheimia sp. LPK (KCTC 10558BP). Next, each of pta-ackA genes and a ppc gene was disrupted from the bacterial mutant Mannheimia sp. LPK, thereby constructing various bacterial mutants. Then, such bacterial mutants were confirmed to produce succuiic acid at high concentration while producing little or no other organic acids.
The inventive bacterial mutants (Mannheimia sp. LPK, LPK4 and LPK7) are facultative anaerobic, gram-negative, non-mobile rods or cocobacilH, do not produce endospores, and can produce succinic acid in anaerobic conditions.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows a process of constructing a vector containing a disrupted IdhA (pMLKO-sacB).
FIG. 2 shows a process of constructing a vector containing a disrupted pfl (pMPKO-sacB).
FIG. 3 s\iows a process of constructing a bacterial mutant (LPK) by disrupting IdhA sadpfl gaies from Mannheimia succiniciproducens 55E.
FIG. 4 is an electrophoresis photograph showing the disruption of IdhA and pfl genes from Mannheimia sp. LPK (M: lambda Hzndlll size marker, lanes 1-3: PCR product LUl & KMl (1.5 kb); lanes 4-6: PCR product LD2 & KM2 (1.7 kb); lanes 7-9; PCR product PUl & CMl (2.2 kb); and lanes 10-12: PCR product PD2 &CM2(1.6kb)).

FIG. 5 shows the culture characteristics of Mannheimia sp. LPK in anaerobic conditions saturated with COj.
FIG. 6 shows a process of constructing vectM: containing a disrupted p/a and ackA (pPTA-sacB).
FIG. 7 is a process of constructing a vector containing a disrupted ppc (pPPC-sacB).
FIG. 8 shows a process of constructmg bacterial mutant LPK7 by disrupting pta and ackA genes fix)m Mannheimia sp. LPK.
FIG. 9 shows a process of constructing bacterial mutant LPK4 by disrupting a/ipc gene from Mannheimia sp. LPK.
FIG. 10 is an electrophoresis photograph showing the disruption of pta and ackA genes from Mannheimia sp. LPK7 (M: 1-kb ladder size marker, lane 1: PCR product P13 & P14 (1.1 kb); and lane 2: PCR product P15 & P16 (1.5 kb)).
FIG. 11 is an electrophoresis photograph showing the disruption of a ppc gene from Mannheimia sp. LPK4 (M: 1-kb ladder size marker; lane 1: PCR product P13 & P17 (1.1 kb); and lane 2: PCR product P15 & P18 (1.5 kb)).
FIG. 12 shows the cultivation characteristics of Mannheimia sp. LPK7 in anaerobic conditions saturated with CO2.
FIG. 13 shows the cultivation characteristics of Mannheimia sp. LPK4 in anaerobic conditions saturated with CO2.

DETAILED DESCRIPTION OF THE INVENTION
The present invention will hereinafter be described in fiirther detail by exan^>les. It will however be obvious to a person skilled in the art that these exan5)Ies are given for illustrative purpose only, and the present invention is not limited to or by the examples.
Particularly, the following examples illustrate only a method comprising disrupting genes &om a genus Mannheimia strain to obtain bacterial mutants and then producing succinic acid at high concentration by these bacterial mutants. However, methods by which bacterial mutants having disruptions of such genes are obtained ftom other rumen bacterial strains, such as genus Actinobacillus and genus Anaerobiospirillum, and succinic acid is produced using the bacterial strains, will also be obvious to a person skilled in the art
Furthermore, the following examples illustrate only a certain medium and culture method. However, the use of other mediums different from, such as whey, com steep liquor (CSL), as described in literatures (Lee et al., Bioprocess Biosyst. Eng., 26:63, 2003; Lee et al., Appl. Microbiol BiotechnoL, 58:663, 2002; Lee et al, Biotechnol Lett.^ 25:111, 2003; Lee et al, Appl Microbiol Biotechnol, 54:23, 2000; Lee et al., Biotechnol Bioeng., 72:41, 2001), and the use of various methods, such as fed-batch culture and continuous culture, will also be obvious to a person skilled in the art.
Example 1: Construction of pMLKO-sacB
In order to disrupt a lactate dehydrogenase gene {IdkA) by homologous recombination, a gene exchange vector was constructed in the following manner. First, the genomic DNA- of UamUieimia sucdniciproducens 55E (KCTC 0769BP), as a tenqjlate, was subjected to PCR using primers set forth in SEQ ED NO: 1 and

SEQ ID NO: 2 below, and then, the obtained PCR fragmait was cut with Sacl and Pstl and introduced into pUClS (New England Biolabs, Inc., Beverly, Mass.), thereby constructing pUC 18-L1.
SEP ID NO: 1-. S"-CAGTGAAGGAGCTCCGTAACGCATCCGCCG (LSI) SEP ID NO: 2: 5"-CTTTATCGAArCTGCAGGCGGTTTCCAAAA (LPl)
rhereafter, the genomic DNA of Mannheimia succiniciproducens 55E, as a emplate, was subjected to PCR using primers set forth in SEQ ID NO: 3 and SEQ DD NO: 4 below, and the resulting PCR fragment was cut with Pstl and HindSl md introduced into the pUC 18-L I, thereby constructing pUCl 8-L 1-L2. SEQ ID NO: 3: S"-GTACTGTAAACTGCAGCTTTCATAGnAGC (LP2) SEQ ID NO: 4: 5"-GCCGAAAGTCAAGCTTGCCGTCGTTTAGTG (LH2)
)UC4K (Pharmacia, Freibui^, Germany) was cut with Pstl, and the resulting anamycin-resistant gene was fiised with pUC18-Ll-L2 cut with Pstl, thereby instructing pUC18-Ll-KmR-L2. A linker set forth in SEQ ID NO; 5 was Dserted into the pUC18-Ll-KniR-L2 cut with Sacl, thereby making a new A&al utting site.
SEP ID NO: 5: 5"-TCTAGAAGCT
"CR on pKmobsacB (Schafer et al. Gene, 145:69, 1994) as a template was crformed using primers set forth in SEQ ID NO: 6 and 7 below, and the resulting "CR product was cut with JCbal and inserted into the above Xbal restriction nzyme site, thereby constructing pMLKO-sacB (FIG. 1).
SEP ID NO: 6: S"-GCTCTAGACCTTCTATCGCCTTCTTGACG (SXF) SEP ID NO: 7: S"-GCTCTAGAGGCTACAAAATCACGGGCGTC (SXR)
Ixample 2: Construction of pMPKO-sacB
1 ordei to disrupt a pyruvate formate-lyase gene (pjt) by homologous

■ecombination, a genetic exchange vector was constructed in the following nanner. A pKmobsacB template containing a sacB gene (Genbank 02730) was jubjected to PCR using primers set forth in SEQ ID NO: 8 and SEQ ID NO: 9 >elow. The resulting sacB product was cut with Pstl and BamSi and inserted nto pUC19 (Stratagene Cloning Systems. La JoUa, Calif.), ihsrehy constructing )UC19-sacB.
SEP ro NO: 8: 5"-AGCGGArCCCCTTCTATCGCCTTCTTGACG (SBG) SEP ID NO: 9: 5"-GTCCTGCAGGGCTACAAAATCACGGGCGTC (SPR)
rhe genomic DNA of Mannheimia succiniciproducens 55E, as a template, was rubjected to PCR using primers set forth in SEQ ID NO: 10 and SEQ ID NO: 11 >elow. The resulting PCR fragment was cut witii BamYQ. and fused with the 3UC19-sacB cut with BamW.^ thereby constructing pUC19-sacB-pfl.
SEC ID NO: 10: 5"-CATGGCGGATCCAGGTACGCTGAnTCGAr (PBl) SEOIDNQ:!!: S"-CAAGGATCCAACGGATAAAGCmTAITATCPB2)
n order to obtain a chloramphenicol-resistant gene, pACYCI84 (New England 3iolabs, Inc., Beverly, Mass.) as a ternplate was subjected to PCR using primers jet forth in SEQ ID NO: 12 and SEQ ID NO: 13 below. The resulting PCR DToduct was cut with Smdl and fused with the pUC19-sacB-pfl cut with BstWQTl, hereby constmcting pMPKO-sacB (FIG 2).
SEP ID NO: 12: SVCTCGAGCCCGGGGTTTAAGGGCACCAAIAA (CTR) SEP ID NO: 13: S"-CTCGAGCCCCGGGCnTGCGCCGAATAAAT (GIF)
Example 3: Construction oj Mannheimia sp. LPK strain
?IG 3 shows a process of constructing a mutant strain (LPK) by disrupting IdhA md pfl genes from Mannheimia succiniciproducens 55E. Mannheimia "succiniciproducens 55E was plated on LB-glucose medium containing 10 g/1 of glucose, and cultured at 37°C for 36 hours. The colony formed was inoculated

in 10 ml of LB-glucose liquid medium, and cultured for 12 hours. The culture broth which had been sufficiently grown was inoculated by 1% in 100 ml of LB-ghicose liquid medium, and cultured in a shaking incubator at 200 ipm and "iT"C.
When the culture broth reached an OD of about 0.2-0.3 after 4-5hours, it was centriiuged at 4*0 and 4000 ipm for 10 minutes to collect cells. Then, the cells were resuspended in 200 nil of 10% glycerol solution at 4°C. The suspension was centriiuged at 4"C and 4000 ipm for 10 minutes, and tiie cells were collected and resuspended in 200 ml of 10% glycerol solution at 4""C, and then centriiuged at 4°C and 4000ipm for 10 miimtes to collect the cells. The cells were suspended in glycerol at a volume ratio of 1:1, to obtain cell concentrate.
The cell concentrate thus obtained was mixed with the genetic exchange vectors pMLKO-sacB and pMPKO-sacB constructed in Stamples I and 2, and then subjected to electroporation under conditions of 1.8 kV, 25 ^F and 200 ohms. 1 ml of LB-glucose hquid medium was added to the electroporated mixture and cultured in a shaking incubator at 37°C and lOOrpm for one hour. The culture broth was plated on LB-glucose soHd medium contaming a suitable antibiotic [Km (final concaitration of 25 lagtail) or Cm (6.8 jig/ml) and cultured at 37°C for 48 hours or more. In order to select a colony ^here only double crossover occurred, ths colonies formed were streaked on LB-sucrose medium (LB medium with IOOg/1 suCTOse) containing Km 25 Hg/ml) or Cm (6.8|ag/ml). Ailer 24 hours, the formed colonies were streaked again on the same plate.
The colony (mutant) formed on the plate were cultured in LB-glucose liquid medium containing an antibiotic, and a genomic DNA was isolated firom the cultured strain by the method described in Rochelle et al. (FEMS Microbiol. Lett., 100:59, 1992). PCR was performed using the isolated mutant genomic DNA as a template, and the PCR product was electrophoresed to confirm the disruption of IdhA and/?/? genes from the PCR product

hi order to confirm the disruption of tiie IdkA gene, PCRs were performed twice m the following manners. First, the mutant genomic DNA as a teai^late was subjected to PCR using primers set forth in SEQ ID NO: 14 and SEQ ID NO: 15. SEC ID NO: 14: S"-GACGnTCCCGTTGAATATGGC (KMl) SEC ID NO: 15: S"-CArTGAGGCGTATTATCAGGAAAC (LUl)
Then, the mutant genomic DNA as a template was subjected to PCR using primers set forth in SEQ ID NO: 16 and SEQ ID NO: 17 below. The products obtained m flie two PCRs were subjected to gel electrophoresis to confirm the disruption of IdhA by their size (1.5 kb) (FIG 4).
SEP ID NO: 16: 5"-GCAGTTTCArTTGArGCTCGArG (KM2) SEP ID NO: 17: 5"-CCTCTrACGArGACGCATCTTTCC (LD2)
In order to confirm the disruption of pfl, PCRs were performed twice in the following manner. First, the mutant genomic DNA as a template was subjected to PCR using primers set forth in SEQ ID NO: 18 and SEQ ID NO: 19 below. SEP ID NO: 18: S"-GGTGGTATATCCAGTGATTTmTCTCCAT (CMl) SEP ID NO: 19:5"-CTTTGCAACATTATGGTATGTATTGCCG (PUI)
Then, the mutant genomic DNA as a template was subjected to PCR using primeis set forth in SEQ ID NO: 20 and SEQ ID NO: 21. The products obtained in tiie two PCRs were subjected to gel electrophoresis to confirm the disruption of pfl by their size (l.Skb) (FIG. 4). In FIG. 4, M represents a Lambda Htndlll size maricer, lanes 1-3 represent Ihe PCR product LUl & KMl (1.5kb), lanes 4-6 represent the PCR product LD2 & KM2 (l.Tkb), lanes 7-9 represent the PCR product PUI & CM.I (2.2kb), and lanes 10-12 represent the PCR product PD2 & CM2 (1.6kb).
SEOIDNO:20:5"-TACTGCGATGAGTGGCAGGGCGGGGCGTAA (CM2) SEP ID NO: 21:5"-CCCCAGCATGTGCAAATCTTCGTCAC (PD2)

The disruption of IdhA was confirmed by the fact that the product resulted fix)m the PCR using the primers (LUl and KMl) of SEQ ID NO: 14 and SEQ ED NO: 15 has a size of 1.5 kb an at tiie same time the product resulted from the PCR using the primers (LD2 and KM2) of SEQ ID NO; 16 and SEQ ID NO: 17 has a size of 1.7 kb. And, the disnqjtion of pfl was confirmed by the fact that the product resulted fixim the PCR using the primers (PUl and CM!) of SEQ ID NO; 18 and SEQ ID NO: 19 has a size of 22 kb and at die same time the product resulted from the PCR using the primers (PD2 and CM2) of SEQ ID NO; 20 and SEQ ID NO: 21 has a size of 1.6 kb. The position of each primer is shown in FIG. 3. The mutant constructed by the above method, i.e., a bacterial mutant having disruptions of IdhA and pjl, was named "Mannheimia sp. LPK" and deposited under accession number KCTC 1088 IBP on November 26, 2003 in the Korean Collection for Type Cultures (KCTC), Korean Research Institute of Bioscience and Biotechnology (KRIBB).
Example 4: Fermentation characteristics of Mannheimia sp. LPK
In order to examine the fermentation charactaistics of Mannheimia sp. LPK constructed in Example 3 above, the mutant was cultured in anaerobic conditions saturated with CO2, and the resulting reaction product was analyzed. First, caibon dioxide was introduced into 100 ml of preculture medium consisting of 20g/L glucose, 5g/L polypeptone, 5g/L yeast extract, 3g/L K2HPO4, Ig/L NaCl, Ig/L (NH4)2S04, 0.2g/L CaClz • 2H2O, 0.2g/L MgClz • 6H2O and lOg/L MgCOa, and then, Mannheimia sp. LPK was inoculated in ttie preculture medium and precultured at 39*C for 14 hours. Then, 0.9 L of culture medium consisting of 20g/L glucose, 5g/L polypeptone, 5g/L yeast extract, 3g/L K2HPO4, Ig/L NaQ, 5g/L (NH4)2S04, 0.2g/L CaQj - 2H2O, 0.2g/L MgClj • 6H2O and 5g/L NajCOa was put in a 2.5-L culture tanV^ and 100 ml of the precultured microorganisms

were inoculated in the culture medium and batch-cultured at SP^C and pH 6.5 while supplying carbon dioxide at a flow rate of 0.25vvm.
The concentration of cells in the culture broth was measured with a spectrophotometer (Ultraspec 3000, Pharmacia Biotech., Sweden), and the amounts of succinate, glucose, lactate, acetate and fonnate were measured by HPLC (Aminex HPX-87H column, Bio-Rad, USA) (FIG. 5). Symbols in FIG- 5, refer to changes in the concentrations of cells (•), succinate (o), glucose (■), formate (O) and acetate (A) with the passage of culture time. As shown in FIG. 5, after 14 hours of culture, the concentration of consumed glucose was 20g/L and the concentration of produced succinate was 17.2g/L, indicating that the yield of succinate (the amount of produced succinate/the amount of consumed glucose) is 31% and the volume productivity of succinate (the concentration of produced niccinate/elapsed time) is 1.23 g/L/h. The inventive mefliod of prwlucing juccinic acid by culturing Mannheimia sp. LPK in anaerobic conditions saturated vith CO2 showed a great increase in yield as compared to tiiat of producing tuccinic acid by culturing parent strain Mannheimia succiniciproducens 55E in maCTobic conditions saturated with CO2, and showed a ratio of succinic acid : icetic acid of 40.7:1, indicating that it can produce succinic acid mth little or no )y-IHX)ducts.
iliample 5: Construction of pPTA-sacB
n order to disrupt a pho^hotransacetylase gene (ptd) and an acetate kinase gene ackA) by homologous recombinatioiL, a genetic exchange vector was constructed Q the following manner- First, the genomic DNA of Mannheimia sp. LPK KCTC 10558BP), as a template, was amplified by PCR using primers set forth in ;EQ ID NO: 22 and SEQ K) NO: 23 below, and the resulting PCR fiagment was ut with Xbal and 5amHI and introduced into pUC19, thereby constructing 1UCI9-PTAI.

SEP ID NO: 22: 5"-GCTCTAGATATCCGCAGTATCACTTTCTGCGC SEP ID NO: 23: S"-TCCGCAGTCGGATCCGGGTTAACCGCACAG
Thereafter, the genomic DNA of Mannheimia sp. LPK as a template was amplified by PCR using primers set forth in SEQ ID NO: 24 and SEQ ID NO: 25 below, and the resulting PCR fragment was cut with Xbal and Sad and introduced into the pUC19-PTAl, thereby constructing pUC19-PTAl2.
SEP ID NP: 24: 5"-GGGGAGCTCGCTAACITAGCrTCTAAAGGCCATGT TTCC SEP ID NO: 25: 5"-GCTCTAGATATCCGGGTCAATATCGCCGCAAC
As a template, plasmid pIC156 (Steimnetz et al. Gene, 142:79, 1994) containing a spcctinomycin-resistant gene (GenBank X02588) was amplified by PCR using primers set forth in SEQ ID NO: 26 and SEQ ID NO: 27 below, and the resulting PCR firagment (spectinomycin-resistant gene) was cut with EcdBN and introduced into the pUC19-PTA12, thereby constructing pUC19-PTAlS2 having the spectinomycin-resistant gene. The constructed pUC19-PTAlS2 was cut with JSQCI and BamHI and introduced into pUC19-SacB (see Example 2), thereby constructing a pPTA-sacB vector (FIG. 6).
SEP ID NP: 26: 5"-GAATTCGAGCTCGCCCGGGGATCGATCCTC
SEP ID NP: 27: 5"-CCCGGGCCGACAGGCTTTGAAGCATGCAAATGTCAC
Example 6: Construction of pPPC-simB
[n order to disrupt a phosphoenolpyruvate carboxylase gene {ppc) by homologous recombination, a genetic exchange vector was constructed in the following [naimer. First, the genomic DNA of Mannheimia sp. LPK, as a template, was unplified by PCR using primers set forth in SEQ ID NO: 28 and SEQ ID NO: 29, md die resulting PCR fragment was cut with Xbal and BamSi and introduced nto pUC19, thereby constructing pUC19-PPCl.

SEP ID NO: 28: S"-TACGGATCCCCAGAAAATCGCCCCCATflTCGA SEP ID NO: 29-. S"-GCTCTAGATATCGTTTGATATTGTTCCGCCACATrTG
Thereafter, the genomic DNA of Mannheimia sp. LPK, as a template, was subjected to PCR using primers set forth in SEQ ID NO: 30 and SEQ ID NO: 31, and the resulting PCR fragment was cut with JOyal and Sad and introduced into the pUC19-PPCl, thereby constractmg pUC19-PPC12.
SEO ID NO: 30:5"-GCTCTAGATATCCGTCAGGAAAGCACCCGCCATAGC SEC ID NO: 31: 5*-GGG A spectinomycin-resistant gene cut with EcoVN (see Example 5) was introduced into the pUC19-PPC12 to conshiict pUC19-PPClS2. The pUC19-PPClS2 was cut with Sad and BamSl and introduced into the pUC19-SacB, thereby constructing a pPPC-sacB vector (FIG. 7).
Example 7: Construction of Mannheimia sp. LPK7 and LPK4 strains
. FIG. 8 and FIG. 9 show processes of constructing mutant strains LPK7 and LPK4 by disrupting pta-ackA. and ppc fii>m Mannheimia sp. LPK, respectively. Mannheimia sp. LPK was plated on LB-glucose medium containing lOgA glucose, and cultured at 3>TC for 36 hours. The colony formed was inoculated in 10 ml LB-glucose hquid medium and cultured for 12 hours. The culture broth which had been sufficiently grown was inoculated by 1% in 100 ml LB-glucose liquid medium and cultured in a shaking incubator at 37*"C.
Cell concentrate was collected from the resulting culture broth in the same manner as descaibed in Example 3. The collected cell concentrate was mixed with the genetic exchange vectors pPTA-sacB and pPPC-sacB constructed in Exanqiles 5 and 6, and then subjected to electroporation under conditions of 1.8 kV, 75°? and 200 ohms. The electroporated mixture was added with 1 ml of

LB-glucose liquid medium and cultured in a shaking incubator at 200 rpm and 37"*C for one hour.
The culture broth was plated on LB-glucose solid mediimi containing a spectinomycin antibiotic (final concentration: 50 (g/ml), and cultured at 37°C for at least 48 hoiurs. In order to select a colony where double crossover occurred, the colonies formed were streaked on LB-sucrose medium (LB mediiun containing 100 g/1 of sucrose) containing 50 (g/ml of spectinomycin. After 24 hours, the formed colonies were re-streaked on the same plate. The colony (mutant) formed on the plate was cultured in LB-glucose hquid medium containing an antibiotic, and a genomic DNA was isolated fiT>m the cultured strain by the method of Rochelle et al. The isolated mutant genomic DNA as a template was anqjlified by PCR, and the PCR product was electrophoresed to confirm the disruption of each of pta-ackA andppc.
To confirm flie disruption of pta-ackA, PCRs were performed twice in the following manner. First, the mutant genomic DNA as a template was subjected to PCR using primers set forfii in SEQ ID NO: 32 and SEQ ID NO: 33 below. Then, the mutant genomic DNA as a ten^late was subjected to PCR using primers set forth in SEQ ID NO: 34 and SEQ ID NO: 35.
SEC ID NO: 32: 5"-CCTGCAGGCATGCAAGCTTGGGCTGCAGGTCGACTC SEP ID NO: 33: S"-GCTGCCAAACAACCGAAAATACCGCAATAAACGGC SEQ ID NO: 34: S"-GGATGTAACnTACTGGATATAGCTAGAAAAGaCATCGaGGAG SEP ID NO: 35: S"-GCAACGCGAGGGTCAATACCGAAGGATTTCGCCG
The products obtained in the two PCRs WCTC subjected to gel electrophoresis to confirm ttie disruption of pta-ackA by their size (FIG. 10). In FIG. 10, M represents a 1-kb ladder size marker, lane 1 rejiresents the PCR product P13 & P14 (1.1 kb), and lane 2 represents the PCR product P15 & P16 (1.5 kb). The disruption of pta-ackA was confirmed fay the fact the product resulted fix>m the

PCR using the primers of SEQ ID NO: 32 and SEQ ID NO: 33 (P13 & P14) has a size of 1.1 kb at flie same time the product resulted from the PCR using the primers of SEQ ID NO: 34 and SEQ ID NO: 35 (P15 & P16) has a size of 1.5 kb. The positions of the primers are shown in FIG. 8. The mutant strain constructed as described above, i.e., a strain resulted from tiie disruption of pta-ackA from Mannheimia sp. LPK, was named "Mannheimia sp. LPK7" and deposited under accession number *"KCTC 10626BP" in KCTC, an international depositary authority.
Furthermore, to confirm the disruption of ppc, PCRs were performed twice in the following msama. First, the mutant genomic DNA as a template was subjected to PCR using primers set forth in SEQ ID NO: 32 and SEQ ID NPO: 36. Then, the mutant genomic DNA as a template was subjected to PCR using primers set forth in SEQ ID NO: 34 and SEQ ID NO: 37.
SEC IP NO: 36: 5"-GATCCAGGGAATGGCACGCAGGCTTrCAACGCCGCC SEC ID NO: 37: S"-GCAAAGCCAGAGGAATGGATGCCATTAACCAATAGCG
The products obtained in the two PCRs were subjected to gel electrophoresis to confirm (he disruption of ppc by flieir size (FIG. 11). In FIG. 11, M represents a 1-kb ladder size marker, lane 1 is the PCR product P13 & P17 (l.lkb), and lane 2 represents the PCR product PIS & P18 (1.5kb). The disruption of ppc was confirmed by the fact that the product resulted from the PCR using the primers of SEQ ID NO: 32 and SEQ ID NO: 36 (P13 & PIT) has a size of 1.1 kb at the same time the product resulted from the PCR using the primflers of SEQ ID NO: 34 and SEQ ID NO: 37 (P15 & P18) has a size of 1.5 kb. The positions of the primers are shown in FIG. 9. The mutant sti^iin constructed as descdbed above, i.e., a strain resulted from the disruption of ppc from Mannheimia sp. LPK, was named "Mannheimia sp. LPK4".
Example 8: Fermentation characteristics of LPK7 and LPK4

hi order to examine the fermentation characteristics of Mannheimia sp. LPK7 and LPK4 constructed in Example 7 above, the mutant strains were cultured m anaerobic conditions saturated with CO2, and the resulting reaction products were aoalyzed. First, carbon dioxide was introduced into 200inl of the preculture mediimi as described in Example 4, and each of Mannheimia sp. LPK7 and LPK4 was inoculated in the precultuxe medium and precultured at 39*C for 24 hours. Next, 1.8 L of a culture medium, which is the same as that in Exan^le 4 except that glucose concentration is 18 g/L (final lOOniNf), was put in a 6.6 L culture tank, and 100 ml of the precultured microorganisms was inoculated in the culture medium and then batch-cultured at 39°C and pH 6.5 while supplying carbon dioxide at a flow rate of 0.25wm.
The concentrations of cells, succinate, glucose, lactate, acetate and formate were measured ia the same mamier as in Example 4 (FIG. 12 and FIG. 13). Symbols in FIG. 12 and FIG. 13 refer to changes in the concMitrations of cells (• m upper portion), succinate (• m lower porticm), glucose (a), formate (♦) and acetate (A) with the passage of culture time. As shown in FIG. 12, after 22 hours of the culture of Mannheimia sp. LPK7, the concentration of consumed glucose was lOOmM and the concentration of produced succinate was 124mM, indicating that the yield of succinate (the amount of produced succinate/the amount of consumed glucose) is 124 mol%. And, the production of acetate was remarkably reduced (Table 1). Also, as shown in FIG. 13, after 22 hours of the culture of Mannheimia sp. LPK4, the concentration of consumed glucose was lOOmM and the concentration of produced succinate was 123.7mM, indicating that the yield of succinate (the amount of produced succinate/the amount of consumed glucose) is 123.7 mol%. And, the production of acetate was greatly reduced as conqsared to tiiat m the wild type (Table 1).

iVhile &e present invention has been described in detail with Teforence to the T>ecific features, it will be ^jparent to persons skilled in the art tiiat fiiis iescription is only for a preferred embodiment and does not limit the scope of the )resent invention. Thus, the substantial scope of the present invention will be iefined by the appended claims and equivalents thereof.

INDUSTRIAL APPLICABILITY
As described and provided above in detail, Mannheimia sp. mutant strains (LPK, LPK7 and LPK4) produce succinic acid in anaerobic conditions saturated with CO2 and are facultative anaerobic strains having high resistance to oxygen. Thus, tiie production of succinic acid using such mutants can not only eliminate the fCTmaitation process instability caused by oxygen exposure, etc., but also eliminate the production of other organic acids, as con^pared to the prior method of producing succinic acid using obligate anaerobic strains, thereby making it possible to cq)timize and maximize a purification process and production yield.

WE CLAIM :
1. A rumen bacterial mutant which a lactate dehydrogenase-encoding gene (IdhA) and a pyruvate formate-lyase-encoding gene (p_/I) have been disrupted, and has the property of producing succinic acid in anaerobic conditions, wherein the rumen bacteria are selected from the group consisting of genus Mannheimia, genus Actinobacillus and genus Anaerobiospirillum.
2. The rumen bacterial mutant as claimed in claim 1, wherein a phosphotransacetylase-encoding gene (pla) and an acetate kinase-encoding gene (ackA) have additionally been disrupted from the said rumen bacteria that a lactate dehydrogenase-encoding gene (IdhA) and a pyruvate formate-lyase-encoding gene (pJT) have been disrupted.
3. The rumen bacterial mutant as claimed in claim I, wherein a phosphoenolpyruvate carboxylase-encoding gene (ppc) has additionally been disrupted from the said rumen bacteria that a lactate dehydrogenase-encoding gene {IdhA) and a pyruvate formate-lyase-encoding gene (pJT) have been disrupted.
4. The rumen bacterial mutant as claimed in any one of claims 1 to 3, wherein the rumen bacteria are homo-fermentative bacteria that produce only succinic acid while producing little or no other organic acids.
5. The rumen bacterial mutant as claimed in claim 1, wherein the rumen bacterial mutant is Mannheimia sp. LPK.
6. The rumen bacterial mutant as claimed in claim 5, wherein said Mannheimia sp. LPK is KCTC 10558BP.
7. The rumen bacterial mutant as claimed in claim 2, wherein the rumen bacterial mutant is Mannheimia sp. LPK7.

8. The rumen bacterial mutant as claimed in claim 7, wherein said Mannheimia sp. LPK7 is KCTC 10626BP.
9. The rumen bacterial mutant as claimed in claim 3, wherein the rumen bacterial mutant is Mannheimia sp. LPK4.
10. A method for producing rumen bacterial mutant that has the property of producing succinic acid in anaerobic conditions, the method comprising disrupting a lactate dehydrogenase-encoding gene (JdhA) and a pyruvate formate-lyase-encoding gene {pfl) fitjm rumen bacteria that are selected from the group consisting of genus Mannheimia, genus Actinobacillus and genus Anaerobiospirillum.
11. The method for producing rumen bacterial mutant as claimed in claim 10, wherein the method comprising additionally disrupting a phosphotransacetylase-encoding gene (pla) and an acetate kinase-encoding gene (ackA) from the said rumen bacteria that a lactate dehydrogenase-encoding gene (JdhA) and a pyruvate formate-lyase-encoding gene (pjl) have been disrupted.
12. The method for producing rumen bacterial mutant as claimed in claim 10, wherein the method comprising additionally disrupting a phosphoenolpyruvate carboxylase-encoding gene (ppc) from the said rumen bacteria that a lactate dehydrogenase-encoding gene (IdhA) and a pyruvate formate-lyase-encoding gene (p/l) have been disrupted.
13. The method for producing the rumen bacterial mutant as claimed in claim 11 or 12, wherein the rumen bacterial mutant having disruptions of a lactate dehydrogenase-encoding gene (IdhA) and a pyruvate formate-lyase-encoding gene (pfl) is Mannheimia sp. LPK (KCTC 10558BP).
14. The method for producing the rumen bacterial mutant as claimed in claim 10, wherein the disruption of the IdhA and pfJ genes is performed by homologous recombination.

15. The method for producing the rumen bacterial mutant as claimed in claim 14, wherein the homologous recombination is perfonned using a genetic exchange vector containing a disrupted idkA and a genetic exchange vector containing a disrupted/?//.
16. The method for producing the rumen bacterial mutant as claimed in claim 15, wherein the genetic exchange vector containing a disrupted IdhA is pMLKO-sacB, and the genetic exchange vector containing a disrupted p/l is pMPKO-sacB.
17. The method for producii^ the rumen bacterial mutant as claimed in claim II, wherein the disruption oflhepla and ackA genes is performed by homologous recombination.
18. The method for producing the rumen bacterial mutant as claimed in claim 17, wherein the homologous recombination is perfonned using a genetic exchange vector containing a disruptedpfa and ackA.
19. The method for producing the rumen bacterial mutant as claimed in claim 18, wherem the genetic exchange vector containing a disrupted pta and ackA is pPTA-sacB.
20. The method for producing the rumen bacterial mutant as claimed in claim 12, wherein the disruption of the ppc gene is performed by homologous recombination.
21. The method for producing the rumen bacterial mutant as claimed in claim 20, wherein the homologous recombination is performed using a genetic exchange vector contaming a disrupted/Tpc.
22. The method for producing the rumen bacterial mutant as claimed in claim 21, wherein the genetic exchange vector containing a disrupted/?/)c is pPPC-sacB.

23. A genetic exchange vector pMLKO-sacB containing a disrupted IdhA.
24. A genetic exchange vector pMPKO-sacB containing a disrupted/?//.
25. A genetic exchange pPTA-sacB containing a disruptedpto and ackA.
26. A genetic exchange vector pPPC-sacB containing a disrupted ppc.
27. A method for producing succinic acid, the method comprising the steps of: culturing the rumen bacterial mutant as claimed in any one claim among claims 1 to 3 in anaerobic condition; and recovering succinic acid from the culture broth.

28. The method for producing succinic acid as claimed in claim 27, wherein the culturing step is homo-fermentation which produce succinic acid.
29. The method for producing succinic acid according to claim 27, wherein the rumen bacterial mutant is Mannheimia sp. LPK, LPK7 or LPK 4.

Documents:

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Patent Number

228001

Indian Patent Application Number

1865/CHENP/2006

PG Journal Number

10/2009

Publication Date

06-Mar-2009

Grant Date

27-Jan-2009

Date of Filing

26-May-2006

Name of Patentee

KOREA ADVANCED INSTITUTE OF SCIENCE AND TECHNOLOGY

Applicant Address

373-1, Guseong-dong, Yuseong-gu, Daejeon 305-701

Inventors:

#	Inventor's Name	Inventor's Address
1	LEE, Sang Yup	EXPO Apt. 212-702, Cheonmin-dong, Yuseong-gu, Daejeon 305-761
2	LEE, Sang Jun	Daewoo Apt. 305-604, Jijok-dong, Yuseong-gu, Daejeon 305-330 ,

PCT International Classification Number

C12N1/21

PCT International Application Number

PCT/KR2004/001210

PCT International Filing date

2004-05-20

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	10-2003-0084934	2003-11-27	Republic of Korea
2	10-2003-0028105	2004-04-23	Republic of Korea