WO2005052135A1

WO2005052135A1 - Novel rumen bacteria variants and process for preparing succinic acid employing the same

Info

Publication number: WO2005052135A1
Application number: PCT/KR2004/001210
Authority: WO
Inventors: Sang Yup Lee; Sang Jun Lee
Original assignee: Korea Advanced Institute Of Science And Technology
Priority date: 2003-11-27
Filing date: 2004-05-20
Publication date: 2005-06-09
Also published as: EP1692271A4; BRPI0416437A; NZ547305A; DE602004022584D1; RU2006122804A; RU2376369C2; EP1692271B2; US7470530B2; AU2004292642A1; DK1692271T3; CA2545363C; US20070054387A1; JP2007512015A; ATE439427T1; EP1692271A1; DK1692271T4; AU2004292642B2; EP1692271B1; CA2545363A1; JP4672671B2

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) (which are involved in the production of lactic acid, formic acid and acetic acid) from rumen bacteria; a novel bacterial mutant (Mannheimia sp. LPK7) having disruptions of a lactate dehydrogenase gene (ldhA), a pyruvate formate-lyase gene (plf), a phosphotransacetylase gene (pta), and a acetate kinase gene (ackA); a novel bacterial mutant (Mannheimia sp. LPK4) having disruptions of a lactate dehydrogenase gene (ldhA), a pyruvate formate-lyase gene (pfl) and a phosphoenolpyruvate carboxylase gene (ppc) involved in the immobilization of CO2 in a metabolic pathway of producing succinic acid; and a method for producing succinic acid, which is characterized by the culture of the above mutants in anaerobic conditions. The inventive bacterial mutants have the property of producing succinic acid at high concentration while producing little or no organic acids, as compared to the prior wild-type strains of producing various organic acids. Thus, the inventive bacterial mutants are useful as strains for the industrial production of succinic acid.

Description

NOVEL RUMEN BACTERIA VARIANTS AND PROCESS FOR PREPARING SUCCINIC ACID EMPLOYING THE SAME

TECHNICAL FIELD

The present invention relates to a rumen bacterial mutant which produce succinic acid at high concentration while producing little or no other organic acids, as well as a method for producing succinic acid, which is characterized by the culture of such mutants in anaerobic conditions.

BACKGROUND ART

Various anaerobic microorganisms, including Succinivibrio dextrinosolvens, Fibrobacter succinogenes, Ruminococcus flavefaciens 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 Anaerobio spirillum succiniciproducens known to produce succinic acid at high concentration and high yield from glucose upon the presence of excessive C0₂ (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 that it has low yield and costs a great deal in a purification process of removing other organic acids except succinic 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 formate-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, other metabolites (acetic acid and ethanol) account for about half of the production of succinic acid. In 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 enzymes, 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 increasing the production of succinic acid (Vemuri et al, Appl Environ. Microbiol, 68:1715, 2002; Millard et al, Appl. Environ. Microbiol, 62:1808, 1996; Chao and Liao, Appl. Environ. Microbiol, 59:4261, 1993; Stols and Donnelly, Appl Environ. Microbiol, 63:2695, 1997).

Meanwhile, it is known that the disruption of ptsG in E. coli contributes to an improvement of bacterial production and succinic acid production (Chatterjee 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 in the case of E. coli. Recently, an attempt is actively conducted in which the genes of enzymes that immobilize C0₂ in a metabolic pathway 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 was so low, as a result of that, it did not reach an industrially applicable level.

DISCLOSURE OF INVENTION

Accordingly, during our extensive studies to develop bacterial strains that produce succinic acid at high yield, the present inventors constructed bacterial mutant Mannheimia 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 of rumen bacteria, and constructed bacterial mutants Mannheimia sp. LPK7 and LPK4, by the disruption of phosphotransacetylase gene (pta) and an acetate kinase gene (ackA), 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 succinic acid at high yield, thereby completing the 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 succinic 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 formate-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 (IdhA), a pyruvate formate-lyase-encoding gene (pfl), a phosphotransacetylase-encoding gene (pta) 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 (IdhA), a pyruvate formate-lyase- encoding gene (pfl), and a phosphoenolpyruvate carboxylase-encoding gene (ppc) 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 the present invention, the rumen bacteria are preferably homo-fermentative bacteria that may be selected from the group consisting of genus Mannheimia, genus Actinobacillus and genus Anaerobiospirillum and produce only succinic acid while producing little or no other organic acids. In a preferred embodiment of the present invention, the rumen bacterial mutant is Mannheimia sp. LPK, LPK7 or LPK4.

In still another aspect, the present invention provides a method for producing rumen bacterial mutant that has the property of producing succinic acid at high concentration while producing little or no other organic acids in anaerobic conditions, the method comprising disrupting 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 Actinobacillus and genus Anaerobiospirillum.

In the inventive method for producing the rumen bacterial mutant, the disruptions of the IdhA and pfl genes are preferably performed by homologous recombination. The homologous recombination is preferably performed using a genetic exchange vector containing a disrupted IdhA and a genetic exchange vector containing a disrupted pfl. Preferably, the vector containing a disrupted IdhA is pMLKO- sacB, and the vector containing a disrupted pfl is pMPKO-sacB.

In yet another aspect, the present invention provides a method for producing rumen bacterial mutant that has the property of producing succinic acid at high concentration while producing little or no other organic acids in anaerobic conditions, the method comprising additionally disrupting a phosphotransacetylase-encoding gene (ptα) and an acetate kinase-encoding gene (αckA) from rumen bacteria that are selected from the group consisting of genus Mannheimia, genus Actinobacillus and genus Anaerobiospirillum, and a lactate dehydrogenase-encoding gene (IdhA) and a pyruvate formate-lyase-encoding gene (pfl) have been disrupted.

The disruptions of the pta and ackA genes are preferably performed by homologous recombination. The homologous recombination is preferably performed using a genetic exchange vector containing a disrupted pta and ackA. The genetic exchange vector containing a disrupted pta and ackA is preferably pPTA-sacB.

In yet another aspect, the present invention provides a method for producing rumen bacterial mutant that has the property of producing succinic acid at high concentration while producing little or no other organic acids in anaerobic conditions, the method comprising additionally disrupting a phosphoenolpyruvate carboxylase-encoding gene (ppc) from rumen bacteria that are selected from the group consisting of genus Mannheimia, genus Actinobacillus and genus Anaerobiospirillum, and a lactate dehydrogenase-encoding gene (IdhA) and a pyruvate formate-lyase-encoding gene (pfl) have been disrupted.

The disruption of the ppc gene is preferably performed by homologous recombination. The homologous recombination is preferably performed using a genetic exchange vector containing a disrupted ppc. The genetic exchange vector containing a disrupted ppc is preferably pPPC-sacB.

In the present invention, the rumen bacterial mutant having disruptions of a lactate dehydrogenase-encoding gene (IdhA) and a pyruvate formate-lyase- encoding gene (pfl) is preferably Mannheimia 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 pfl; a genetic exchange vector pPTA-sacB containing a disrupted pta and ackA; and a genetic exchange vector pPPC-sacB containing a disrupted ppc.

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 term "disruption" means that the genes encoding the enzymes are modified such that the enzymes cannot be produced.

In the present invention, each of the 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, thereby 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 succinic 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 cocobacilli, 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 shows a process of constructing a bacterial mutant (LPK) by disrupting IdhA dpfl genes from Mannheimia succiniciproducens 55E.

FIG. 4 is an electrophoresis photograph showing the disruption of IdhA and pfl genes from Mannheimia sp. LPK (M: lambda Hinάlll size marker; lanes 1-3: PCR product LU1 & KM1 (1.5 kb); lanes 4-6: PCR product LD2 & KM2 (1.7 kb); lanes 7-9: PCR product PU1 & CM1 (2.2 kb); and lanes 10-12: PCR product PD2 & CM2 (1.6 kb)). FIG. 5 shows the culture characteristics of Mannheimia sp. LPK in anaerobic conditions saturated with C0₂.

FIG. 6 shows a process of constructing vector containing a disrupted pta 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 constructing bacterial mutant LPK7 by disrupting pta and ackA genes from Mannheimia sp. LPK.

FIG. 9 shows a process of constructing bacterial mutant LPK4 by disrupting a ppc 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 C0₂.

FIG. 13 shows the cultivation characteristics of Mannheimia sp. LPK4 in anaerobic conditions saturated with C0₂. DETAILED DESCRIPTION OF THE INVENTION

The present invention will hereinafter be described in further detail by examples. It will however be obvious to a person skilled in the art that these examples 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 from 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 from 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, corn 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 (IdhA) by homologous recombination, a gene exchange vector was constructed in the following manner. First, the genomic DNA of Mannheimia succiniciproducens 55E (KCTC 0769BP), as a template, was subjected to PCR using primers set forth in SEQ ID NO: 1 and SEQ ID NO: 2 below, and then, the obtained PCR fragment was cut with Sad and Pstl and introduced into pUC18 (New England Biolabs, Inc., Beverly, Mass.), thereby constructing pUCl 8-L1. SEO ID NO: !: 5'-CAGTGAAGGAGCTCCGTAACGCATCCGCCG (LSI) SEQ ID NO: 2: 5'-CTTTATCGAATCTGCAGGCGGTTTCCAAAA (LP1)

Thereafter, the genomic DNA of Mannheimia succiniciproducens 55E, as a template, was subjected to PCR using primers set forth in SEQ ID NO: 3 and SEQ ID NO: 4 below, and the resulting PCR fragment was cut with Pstl and H dIII and introduced into the pUC18-Ll, thereby constructing pUC18-Ll-L2. SEQ ID NO: 3: 5'-GTACTGTAAACTGCAGCTTTCATAGTTAGC (LP2) SEQ ID NO: 4: 5'-GCCGAAAGTCAAGCTTGCCGTCGTTTAGTG (LΗ2)

pUC4K (Pharmacia, Freiburg, Germany) was cut with Pstl, and the resulting kanamycin-resistant gene was fused with pUC18-Ll-L2 cut with Pstl, thereby constructing ρUC18-Ll-KmR-L2. A linker set forth in SEQ ID NO: 5 was inserted into the pUC18-Ll-KmR-L2 cut with Sacl, thereby making a new Xbal cutting site. SEQ ID NO: 5: 5'-TCTAGAAGCT

PCR on pKmobsacB (Schafer et al, Gene, 145:69, 1994) as a template was performed using primers set forth in SEQ ID NO: 6 and 7 below, and the resulting PCR product was cut with Xbal and inserted into the above Xbal restriction enzyme site, thereby constructing pMLKO-sacB (FIG. 1). SEQ ID NO: 6: 5 '-GCTCTAGACCTTCTATCGCCTTCTTGACG (SXF) SEQ ID NO: 7: 5'-GCTCTAGAGGCTACAAAATCACGGGCGTC (SXR)

Example 2; Construction of pMPKO-sacB

In order to disrupt a pyruvate formate-lyase gene (pfl) by homologous recombination, a genetic exchange vector was constructed in the following manner. A pKmobsacB template containing a sacB gene (Genbank 02730) was subjected to PCR using primers set forth in SEQ ID NO: 8 and SEQ ID NO: 9 below. The resulting sacB product was cut with Pstl and BamRl and inserted into pUC19 (Stratagene Cloning Systems. La Jolla, Calif), thereby constructing pUC19-sacB. SEQ ID NO: 8: 5'-AGCGGATCCCCTTCTATCGCCTTCTTGACG (SBG) SEQ ID NO: 9: 5'-GTCCTGCAGGGCTACAAAATCACGGGCGTC (SPR)

The genomic DNA of Mannheimia succiniciproducens 55E, as a template, was subjected to PCR using primers set forth in SEQ ID NO: 10 and SEQ ID NO: 11 below. The resulting PCR fragment was cut with BamRl and fused with the pUC19-sacB cut with BamRl, thereby constructing pUC19-sacB-pfl. SEQ ID NO: 10: 5'-CATGGCGGATCCAGGTACGCTGATTTCGAT (PB1) SEQ ID NO: 11: 5'-CAAGGATCCAACGGATAAAGCTTTTATTAT (PB2)

In order to obtain a chloramphenicol-resistant gene, pACYC184 (New England Biolabs, Inc., Beverly, Mass.) as a template was subjected to PCR using primers set forth in SEQ ID NO: 12 and SEQ ID NO: 13 below. The resulting PCR product was cut with Smal and fused with the pUC19-sacB-pfl cut with iføll07I, thereby constructing pMPKO-sacB (FIG. 2). SEQ ID NO: 12: 5'-CTCGAGCCCGGGGTTTAAGGGCACCAATAA (CTR) SEQ ID NO: 13: 5'-CTCGAGCCCCGGGCTTTGCGCCGAATAAAT (CTF)

Example 3: Construction of Mannheimia sp. LPK strain

FIG. 3 shows a process of constructing a mutant strain (LPK) by disrupting IdhA and 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 n 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- glucose liquid medium, and cultured in a shaking incubator at 200 rpm and 37°C.

When the culture broth reached an OD of about 0.2-0.3 after 4~5hours, it was centrifuged at 4°C and 4000 rpm for 10 minutes to collect cells. Then, the cells were resuspended in 200 ml of 10% glycerol solution at 4°C. The suspension was centrifuged at 4°C and 4000 rpm for 10 minutes, and the cells were collected and resuspended in 200 ml of 10% glycerol solution at 4°C, and then centrifuged at 4°C and 4000rpm for 10 minutes 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 Examples 1 and 2, and then subjected to electroporation under conditions of 1.8 kV, 25 μF and 200 ohms. 1 ml of LB-glucose liquid medium was added to the electroporated mixture and cultured in a shaking incubator at 37°C and 200rpm for one hour. The culture broth was plated on LB-glucose solid medium containing a suitable antibiotic [Km (final concentration of 25 μg/ml) or Cm (6.8 μg/ml) and cultured at 37°C for 48 hours or more. In order to select a colony where only double crossover occurred, the colonies formed were streaked on LB-sucrose medium (LB medium with lOOg/1 sucrose) containing Km 25 μg/ml) or Cm (6.8μg/ml). After 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 from 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 pfl genes from the PCR product. In order to confirm the disruption of the IdhA gene, PCRs were performed twice in the following manners. First, the mutant genomic DNA as a template was subjected to PCR using primers set forth in SEQ ID NO: 14 and SEQ ID NO: 15. SEQ ID NO: 14: 5 '-GACGTTTCCCGTTGAATATGGC (KMl) SEQ ID NO: 15: 5'-CATTGAGGCGTATTATCAGGAAAC (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 in the two PCRs were subjected to gel electrophoresis to confirm the disruption of IdhA by their size (1.5 kb) (FIG. 4). SEQ ID NO: 16: 5'-GCAGTTTCATTTGATGCTCGATG (KM2) SEQ ID NO: 17: 5'-CCTCTTACGATGACGCATCTTTCC (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. SEQ ID NO: 18: 5'-GGTGGTATATCCAGTGATTTTTTTCTCCAT (CM1) SEQ LD NO: 19: 5'-CTTTGCAACATTATGGTATGTATTGCCG (PU1)

Then, the mutant genomic DNA as a template was subjected to PCR using primers set forth in SEQ ID NO: 20 and SEQ ID NO: 21. The products obtained in the two PCRs were subjected to gel electrophoresis to confirm the disruption of pfl by their size (1.5kb) (FIG. 4). In FIG. 4, M represents a Lambda H dIII size marker, lanes 1-3 represent the PCR product LUl & KMl (1.5kb), lanes 4-6 represent the PCR product LD2 & KM2 (1.7kb), lanes 7-9 represent the PCR product PU1 & CM1 (2.2kb), and lanes 10-12 represent the PCR product PD2 & CM2 (1.6kb). SEQ IDNO: 20: 5'-TACTGCGATGAGTGGCAGGGCGGGGCGTAA (CM2) SEQ ID NO: 21: 5'-CCCCAGCATGTGCAAATCTTCGTCAC (PD2) The disruption of IdhA was confirmed by the fact that the product resulted from the PCR using the primers (LUl and KMl) of SEQ ID NO: 14 and SEQ ID NO: 15 has a size of 1.5 kb an at the 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 disruption of pfl was confirmed by the fact that the product resulted from the PCR using the primers (PU1 and CM1) of SEQ ID NO: 18 and SEQ ID NO: 19 has a size of 2.2 kb and at the 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 pfl, 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 characteristics 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, carbon dioxide was introduced into 100 ml of preculture medium consisting of 20g/L glucose, 5g/L polypeptone, 5g/L yeast extract, 3g/L K₂HPO₄, lg/L NaCl, lg/L (NH₄)₂SO₄, 0.2g/L CaCl₂ ^• 2H₂O, 0.2g/L MgCl₂ ^• 6H₂0 and lOg/L MgC0₃, and then, Mannheimia sp. LPK was inoculated in the 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, lg/L NaCl, 5g/L (NH₄)₂SO₄, 0.2g/L CaCl₂ ^• 2H₂O, 0.2g/L MgCl₂ ^■ 6H₂0 and 5gL Na₂C0₃ was put in a 2.5-L culture tank, and 100 ml of the precultured microorganisms were inoculated in the culture medium and batch-cultured at 39°C and pH 6.5 while supplying carbon dioxide at a flow rate of 0.25wm.

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 formate 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 (Δ) 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 81% and the volume productivity of succinate (the concentration of produced succinate/elapsed time) is 1.23 g/L/h. The inventive method of producing succinic acid by culturing Mannheimia sp. LPK in anaerobic conditions saturated with C0₂ showed a great increase in yield as compared to that of producing succinic acid by culturing parent strain Mannheimia succiniciproducens 55E in anaerobic conditions saturated with C0₂, and showed a ratio of succinic acid : acetic acid of 40.7:1, indicating that it can produce succinic acid with little or no by-products.

Example 5: Construction of pPTA-sacB

In order to disrupt a phosphotransacetylase gene (pta) and an acetate kinase gene (ackA) by homologous recombination, a genetic exchange vector was constructed in 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 SEQ ID NO: 22 and SEQ ID NO: 23 below, and the resulting PCR fragment was cut with Xbal and BamRl and introduced into pUC19, thereby constructing pUC19-PTAl. SEQ ID NO: 22: 5'-GCTCTAGATATCCGCAGTATCACTTTCTGCGC SEQ IDNO: 23: 5'-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 Sacl and introduced into the ρUC19-PTAl, thereby constructing ρUC19-PTA12. SEQ ID NO: 24: 5'-GGGGAGCTCGCTAACTTAGCTTCTAAAGGCCATGT TTCC SEQ ID NO: 25: 5 '-GCTCTAGATATCCGGGTCAATATCGCCGCAAC

As a template, plasmid pIC156 (Steinmetz et al, Gene, 142:79, 1994) containing a spectinomycin-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 fragment (spectinomycin-resistant gene) was cut with EcoRV and introduced into the ρUC19-PTA12, thereby constructing pUC19-PTAlS2 having the spectinomycin-resistant gene. The constructed pUC 19-PTA1 S2 was cut with Sacl and BamRl and introduced into pUC19-SacB (see Example 2), thereby constructing a pPTA-sacB vector (FIG. 6). SEQ ID NO: 26: 5'-GAATTCGAGCTCGCCCGGGGATCGATCCTC SEQ IDNO: 27: 5'-CCCGGGCCGACAGGCTTTGAAGCATGCAAATGTCAC

Example 6: Construction of pPPC-sacB

In order to disrupt a phosphoenolpyruvate carboxylase gene (ppc) by homologous recombination, a genetic exchange vector was constructed in the following manner. First, the genomic DNA of Mannheimia sp. LPK, as a template, was amplified by PCR using primers set forth in SEQ ID NO: 28 and SEQ ID NO: 29, and the resulting PCR fragment was cut with Xbal and BamRl and introduced into pUC19, thereby constructing ρUC19-PPCl. SEQ ID NO: 28: 5 '-TACGGATCCCCAGAAAATCGCCCCCATGCCGA SEQ ID NO: 29: 5 '-GCTCTAGATATCGTTTGATATTGTTCCGCCACATTTG

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 Xbal and Sacl and introduced into the pUC19-PPCl, thereby constructing pUC19-PPC12. SEQIDNO: 30: 5'-GCTCTAGATATCCGTCAGGAAAGCACCCGCCATAGC SEQ ID NO: 31: 5'-GGGGAGCTCGTGTGGCGCTGCGGAAGTAAGGCAAAAATC

A spectinomycin-resistant gene cut with EcoRV (see Example 5) was introduced into the pUC 19-PPC 12 to construct pUC 19-PPC 1 S2. The pUC 19-PPC 1 S2 was cut with Sacl and BamRl 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 from Mannheimia sp. LPK, respectively. Mannheimia sp. LPK was plated on LB-glucose medium containing lOg/1 glucose, and cultured at 37°C for 36 hours. The colony formed was inoculated in 10 ml LB-glucose liquid 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 described in Example 3. The collected cell concentrate was mixed with the genetic exchange vectors pPTA-sacB and pPPC-sacB constructed in Examples 5 and 6, and then subjected to electroporation under conditions of 1.8 kV, 25°F 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 medium containing a spectinomycin antibiotic (final concentration: 50 (g/ml), and cultured at 37°C for at least 48 hours. In order to select a colony where double crossover occurred, the colonies formed were streaked on LB-sucrose medium (LB medium 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 liquid medium containing an antibiotic, and a genomic DNA was isolated from the cultured strain by the method of Rochelle et al. The isolated mutant genomic DNA as a template was amplified by PCR, and the PCR product was electrophoresed to confirm the disruption of each of pta-ackA and ppc.

To confirm the 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 forth in SEQ ID NO: 32 and SEQ ID NO: 33 below. 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: 35. SEQ ID NO: 32: 5 '-CCTGCAGGCATGCAAGCTTGGGCTGCAGGTCGACTC SEQ ID NO: 33: 5*-GCTGCCAAACAACCGAAAATACCGCAATAAACGGC SEQ ID NO: 34: 5'-GCATGTAACTTTACTGGATATAGCTAGAAAAGGCATCGGGGAG SEQ ID NO: 35: 5 '-GCAACGCGAGGGTCAATACCGAAGGATTTCGCCG

The products obtained in the two PCRs were subjected to gel electrophoresis to confirm the disruption of pta-ackA by their size (FIG. 10). In FIG. 10, M represents a 1-kb ladder size marker, lane 1 represents the PCR product PI 3 & P14 (1.1 kb), and lane 2 represents the PCR product P15 & P16 (1.5 kb). The disruption of pta-ackA was confirmed by the fact the product resulted from 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 the 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 the 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 manner. 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. SEQ ID NO: 36: 5'-GATCCAGGGAATGGCACGCAGGCTTTCAACGCCGCC SEQ ID NO: 37: 5'-GCAAAGCCAGAGGAATGGATGCCATTAACCAATAGCG

The products obtained in the two PCRs were subjected to gel electrophoresis to confirm the disruption of ppc by their 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 PI 5 & PI 8 (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 & P17) has a size of 1.1 kb at the same time the product resulted from the PCR using the primers 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 strain constructed as described 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 In order to examine the fermentation characteristics of Mannheimia sp. LPK7 and LPK4 constructed in Example 7 above, the mutant strains were cultured in anaerobic conditions saturated with C0₂, and the resulting reaction products were analyzed. First, carbon dioxide was introduced into 200ml of the preculture medium as described in Example 4, and each of Mannheimia sp. LPK7 and LPK4 was inoculated in the preculture medium and precultured at 39°C for 24 hours. Next, 1.8 L of a culture medium, which is the same as that in Example 4 except that glucose concentration is 18 g/L (final lOOmM), 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 0.25 wm.

The concentrations of cells, succinate, glucose, lactate, acetate and formate were measured in the same manner as in Example 4 (FIG. 12 and FIG. 13). Symbols in FIG. 12 and FIG. 13 refer to changes in the concentrations of cells (• in upper portion), succinate (• in lower portion), glucose (D), 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 compared to that in the wild type (Table 1). The inventive method of producing succinic acid by culturing Mannheimia sp. LPK7 in anaerobic conditions saturated with C0₂ showed a great increase in the yield of succinic acid and also a 9.8 times increase in the ratio of succinic acid: acetic acid, as compared to that of producing succinic acid by culturing parent strain Mannheimia succiniciproducens 55E in anaerobic conditions saturated with C0₂, indicating that the inventive method can produce succinic acid with producing little or no byproducts (Table 1).

As reported by Bulter et al., even if acetate-producing genes in microorganisms known till now are all disrupted, a significant amount of acetate is produced in amino acid and fatty acid metabolisms which are still not established (Bulter et al. PNAS, 101 :2299, 2004). Thus, the present invention cut off all acetate production pathways known till now, and achieved succinate fermentation at high yield and concentration.

Table 1: Comparison of products from fermentation of LPK4 and LPK7 and product from fermentation of 55E in anaerobic conditions

While the present invention has been described in detail with reference to the specific features, it will be apparent to persons skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined 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 C0₂ and are facultative anaerobic strains having high resistance to oxygen. Thus, the production of succinic acid using such mutants can not only eliminate the fermentation process instability caused by oxygen exposure, etc., but also eliminate the production of other organic acids, as compared to the prior method of producing succinic acid using obligate anaerobic strains, thereby making it possible to optimize and maximize a purification process and production yield.

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS F R THE PURPOSE OF PATENT FROCEDURE ? INTERNATIONAL FORM

RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Rule 7.1 : IEE, Sang Yup Korea Advanced Institute of Science and Technology, #373-1, Ruseαng-dong, Yuseong-gu, Daejeon 30&-7O1, Republic of Korea I . IDENTiπCATION OF THE MICROORGANISM

Identification reference given by the Accession number given by the DEPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY:

Mannheimia sp. I-PK KCTC 10553BJP

π. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION

The microorganism identified under I above was accompanied by.'

[ X ] a scientific description

E ] a proposed taxonomic designation

(Mark with a cross where applicable)

IH. RECEIPT AND ACCEPTANCE

This International Depositary Authority accepts the microorganism identified under I above, which was received by it on November 26 2003.

IV. RECEIPT OF REQUEST FOR CONVERSION

The microorganism identified under I above was received by this Internationa] Depositary Authority on and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on

V. INTERNATIONAL DEPOSITARY AUTHORITY

Name: Korean Collection for Type Cultures Signature(s) of ρerson(s) havin the ower

Address: Korea Research Institute of Bioscaence and Biotechnology (KRIBB) #52, Oun~dong, Yusong-ku, Taejon 305-333,

Republic of Korea Date: November 28 2003 BP/4 (KCTC Form 17) sole page INTERNATIONAL FORM RECEIPT IN TBE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Rule 7.1

'IX) : LEE. Sang Yup Korea Advanced Institute of Science and Technology, s373-l, Kuseong-dong. Yuseong-ku, Daejeon 306-701, Republic: of Korea

, I . IDENTIFICATION OF THE MICROORGANISM Identification reference given by the Accession number given by the DEPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY: Mannheimia sp. LPK7 KCTC 10626BP π . SCIENTIFIC DESCRIΓΠON AND/OR PROPOSED TAXONOMIC DESIGNATION The microorganism identified under I above was accompanied by: f x J a scientific description i, ] a proposed taxonomic designation l.Mark with a cross where applicable) rπ. RECEIPT AND ACCEPTANCE This International Depositary Authority accepts the microorganism identified under I above, which was received by it on April 22 2004. . IV. RECEIPT QK REQUEST FOR CONVERSION The microorganism identified under I above was received by this International Depositary .Authority on and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on V. INTERNATIONAL DEPOSITARY AUTHORITY Name: Korean Collection for Type Cultures Signature(s) of persoπ(s) having the power to represent the International Depositary Authority of authorized officialise Address: Korea Research Institute of Bioscience and Biotechnology (KRIBB) =.72. Oun-dong, Yusong- u, Tiicion 305 -33.^'!. PΛRl YoιiB-Ha Director Republic of Korea Date: April 27 2004

Claims

THE CLAIMS

What is Claimed is: A rumen bacterial mutant which a lactate dehydrogenase-encoding gene (IdhA) and a pyruvate formate-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.

2. A rumen bacterial mutant which a lactate dehydrogenase-encoding gene (IdhA), a pyruvate formate-lyase-encoding gene (pfl), a phosphotransacetylase- encoding gene (pta) 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.

3. A rumen bacterial mutant which a lactate dehydrogenase-encoding gene (IdhA), a pyruvate formate-lyase-encoding gene (pfl), and a phosphoenolpyruvate carboxylase-encoding gene (ppc) 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.

4. The rumen bacterial mutant according to any one claim among claims 1-3, wherein the rumen bacteria are selected from the group consisting of genus Mannheimia, genus Actinobacillus and genus Anaerobiospirillum.

5. The rumen bacterial mutant according to any one claim among claims 1-3, wherein the rumen bacteria are homo-fermentative bacteria that produce only succinic acid while producing little or no other organic acids.

6. The ramen bacterial mutant according to claim 1, wherein the rumen bacterial mutant is Mannheimia sp. LPK.

7. The rumen bacterial mutant according to claim 6, wherein said Mannheimia sp. LPK is KCTC 10558BP.

8. The rumen bacterial mutant according to claim 2, wherein the rumen bacterial mutant is Mannheimia sp. LPK7.

9. The rumen bacterial mutant according to claim 8, wherein said Mannheimia sp. LPK7 is KCTC 10626BP.

10. The rumen bacterial mutant according to claim 3, wherein the rumen bacterial mutant is Mannheimia sp. LPK4.

11. A method for producing rumen bacterial mutant that has the property of producing succinic acid at high concentration while producing little or no other organic acids in anaerobic conditions, the method comprising disrupting 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 Actinobacillus and genus Anaerobiospirillum.

12. A method for producing ramen bacterial mutant that has the property of producing succinic acid at high concentration while producing little or no other organic acids in anaerobic conditions, the method comprising additionally disrapting a phosphotransacetylase-encoding gene (pta) and an acetate kinase- encoding gene (ackA) from rumen bacteria that are selected from the group consisting of genus Mannheimia, genus Actinobacillus and genus Anaerobiospirillum, and a lactate dehydrogenase-encoding gene (IdhA) and a pyruvate formate-lyase-encoding gene (pfl) have been disrupted.

13. A method for producing ramen bacterial mutant that has the property of producing succinic acid at high concentration while producing little or no other organic acids in anaerobic conditions, the method comprising additionally disrapting a phosphoenolpyruvate carboxylase-encoding gene (ppc) from rumen bacteria that are selected from the group consisting of genus Mannheimia, genus Actinobacillus and genus Anaerobiospirillum, and a lactate dehydrogenase- encoding gene (IdhA) and a pyruvate formate-lyase-encoding gene (pfl) have been disrapted.

14. The method for producing the ramen bacterial mutant according to claim 12 or 13, wherein the ramen 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).

15. The method for producing the rumen bacterial mutant according to claim 11 , wherein the disruption of the IdhA and pfl genes is performed by homologous recombination.

16. The method for producing the ramen bacterial mutant according to claim 15, wherein the homologous recombination is performed using a genetic exchange vector containing a disrapted IdhA and a genetic exchange vector containing a disrapted pfl.

17. The method for producing the rumen bacterial mutant according to claim 16, wherein the genetic exchange vector containing a disrapted IdhA is pMLKO-sacB, and the genetic exchange vector containing a disrapted pfl is pMPKO-sacB.

18. The method for producing the ramen bacterial mutant according to claim 12, wherein the disruption of the pta and acJA genes is performed by homologous recombination.

19. The method for producing the ramen bacterial mutant according to claim 18, wherein the homologous recombination is performed using a genetic exchange vector containing a disrupted pta and ackA.

20. The method for producing the ramen bacterial mutant according to claim 19, wherein the genetic exchange vector containing a disrupted pta and ackA is pPTA-sacB.

21. The method for producing the ramen bacterial mutant according to claim 13 , wherein the disruption of the ppc gene is performed by homologous recombination.

22. The method for producing the ramen bacterial mutant according to claim 21 , wherein the homologous recombination is performed using a genetic exchange vector containing a disrapted ppc.

23. The method for producing the ramen bacterial mutant according to claim 22, wherein the genetic exchange vector containing a disrapted ppc is pPPC-sacB.

24. A genetic exchange vector pMLKO-sacB containing a disrupted IdhA.

25. A genetic exchange vector pMPKO-sacB containing a disrupted/?/?.

26. A genetic exchange pPTA-sacB containing a disrupted pta and ackA.

27. A genetic exchange vector pPPC-sacB containing a disrapted ppc.

28. A method for producing succinic acid, the method comprising the steps of: culturing the ramen bacterial mutant of any one claim among claims 1-3 in anaerobic condition; and recovering succinic acid from the culture broth.

29. The method for producing succinic acid according to claim 28, wherein the culturing step is homo-fermentation which produce succinic acid at high concentration while producing little or no other organic acids.

30. The method for producing succinic acid according to claim 28, wherein the ramen bacterial mutant is Mannheimia sp. LPK, LPK7 or LPK 4.