Abstract:
A nucleotide sequence encoding a malic enzyme and a method for preparing succinic acid using the same, more particularly, a maeB nucleotide sequence encoding a malic enzyme B having the activity of converting pyruvic acid or pyruvate to malic acid or malate, or vice versa, a recombinant vector containing the gene, a microorganism transformed with the recombinant vector, and a method for preparing succinic acid using the transformed microorganism.

Description:
BACKGROUND OF THE INVENTION  
       [0001]     1. Field of the Invention  
         [0002]     The present invention relates to a novel gene encoding a malic enzyme and a method for preparing succinic acid using the same, more particularly, to a maeB gene encoding a malic enzyme B having the activity of converting pyruvic acid or pyruvate to malic acid or malate, or vice versa, a recombinant vector containing the gene, a microorganism transformed with the recombinant vector, and a method for preparing succinic acid using the transformed microorganism.  
         [0003]     2. Background of the Related Art  
         [0004]     Succinic acid, which is a dicarboxylic acid (HOOCCH 2 CH 2 COOH) with four carbon atoms initially purified from amber resin, is used in a very wide range of industrial applications (Zeikus et al.,  Appl. Microbiol. Biotechnol.,  51:545, 1999). Particularly, as the utility of succinic acid as a main raw material of biodegradable polymers was recently proven, a rapid increase in the demand of succinic acid is expected (Willke et al.,  Appl. Microbiol. Biotechnol.,  66:131, 2004).  
         [0005]     Succinic acid can be produced by chemical synthesis and fermentation. Most commercially available succinic acid recently has been produced from n-butane as a starting material derived from LNG or crude petroleum, by chemical manufacturers, such as BASF, DuPont and BP Chemicals. Chemical processes for the synthesis of succinic acid have the problem that they cause the discharge of large amounts of harmful solid wastes, waste solutions and waste gases (including carbon monoxide) during the preparation of succinic acid, and particularly, have the limitation that they use fossil raw material as a basic material. Only a small amount of succinic acid, which is used in special applications, such as medical drugs, is currently produced by traditional microbial processes.  
         [0006]     In an attempt to solve the described problems occurring in the chemical processes for the synthesis of succinic acid, studies on the production of succinic acid by fermentation processes have been conducted by many researchers. The method for the production of succinic acid by fermentation is a method of producing succinic acid from renewable raw materials using microorganisms. Bacterial strains which are used in the production of succinic acid can be broadly divided into recombinant  E. coli  and ruminal bacteria, such as  Actinobacillus, Anaerobiospirillum, Bacteroides, Mannheimia, Succinimonas, Succinivibrio,  etc.  
         [0007]     A research team of the University of Chicago has attempted to increase the production of succinic acid by preparing a mutant strain AFP111 (ATCC No. 202021) in which  E. coli  ldh and pfl genes involved in the production of lactic acid and formic acid have been removed and a ptsG gene of the glucose transfer system has been manipulated (U.S. Pat. No. 5,770,435).  
         [0008]     Among ruminal bacteria,  Actinobacillus, Anaerobiospirillum  and  Mannheimia  strains have been relatively much-studied. Michigan Biotechnology Institute (MBI) has developed an  Actinobacillus succinogenes  130Z strain (ATCC No. 55618) and a process for producing a high concentration of succinic acid using the same (U.S. Pat. No. 5,504,004). Also, such institute has developed  Anaerobiospirillum succiniciproducens  and its mutant strains, and a process for the production and purification of succinic acid (U.S. Pat. Nos. 5,521,075; 5,168,055; and 5,143,834).  
         [0009]     However, the processes for preparing succinic acid using the described strains have low productivity and result in the production of large amounts of byproducts in addition to succinic acid, thus requiring high costs for the separation and purification of succinic acid. Accordingly, there has been an urgent need for the development of a bacterial system that has high productivity and at the same time, can inhibit the production of byproducts (Hong et al.,  Biotechnol. Lett.,  22:871, 2000).  
         [0010]     For this purpose, the isolation of an excellent succinic acid-producing bacterial strain, the establishment of genome sequences and the understanding of metabolic characteristics of bacterial strains based on them are first required. With such basis, it then is necessary to secure gene manipulation technologies required for the construction of a novel gene recombinant bacterial strain. Although there has been a prior attempt to increase the production of succinic acid using the phosphoenolpyruvate carboxykinase (pckA) gene of  Anaerobiospirillum succiniciproducens  (Laivenieks et al.,  Appl. Environ. Microbiol.,  63:2273, 1997), the art has failed to develop a gene recombinant strain based on the full genome sequence of ruminal bacteria.  
         [0011]     Meanwhile, the present inventors previously isolated a  Mannheimia succiniciproducens  MBEL55E strain from the rumen of a Korean cow that produces succinic acid in high efficiency using various substrates, and reported the full genome sequence of the strain (Hong et al.,  Nature Biotechnol.,  22:1275, 2004). Particularly, the above strain is characterized by immobilizing carbon dioxide, known as a greenhouse gas, in the synthesis of succinic acid. Also, this applicant previously prepared succinic acid with high yield by deleting a lactic acid dehydrogenase gene (ldhA) and a pyruvate formate-lyase (pfl) from  Mannheimia succiniciproducens  MBEL55E, so as to construct mutant strain  Mannheimia  sp. LPK (KCTC 10558BP), and deleting a phosphotransacetylase gene (pta) and an acetate kinase gene (ackA) from the LPK strain to construct mutant strains  Mannheimia  sp. LPK7, and then culturing the resulting mutant strain in an anaerobic condition (WO 05/052135 A1). However, the mutant strain has a problem that it results in the accumulation of malate to a certain degree as a byproduct during the culture thereof.  
         [0012]     Accordingly, there continues to be an urgent need in the art for the development of a bacterial system for high productivity, low byproduct succinic acid production that overcomes the deficiencies of the prior art  
       SUMMARY OF THE INVENTION  
       [0013]     The present invention relates to a novel gene encoding a malic enzyme B derived from  Mannheimia succiniciproducens  MBEL55E that is usefully employed in the production of succinic acid.  
         [0014]     The present invention relates on one aspect to a recombinant vector containing said gene, and a recombinant microorganism transformed with said recombinant vector.  
         [0015]     Still another aspect of the present invention relates to a method for preparing succinic acid using said recombinant microorganism.  
         [0016]     In one aspect, the present invention relates to a malic enzyme B having an amino acid sequence of SEQ ID NO: 4, which has the activity of either converting pyruvate to malate or converting malate to pyruvate, as well as a gene (maeB) encoding the malic enzyme B. In one preferred aspect of the present invention, said gene preferably has a DNA sequence of SEQ ID NO: 3.  
         [0017]     In another aspect, the present invention relates to a recombinant vector containing the maeB gene and a recombinant microorganism obtained by introducing the maeB gene or the recombinant vector into a host cell selected from the group consisting of bacteria, yeast and mold.  
         [0018]     In a still further aspect of the present invention, the recombinant vector is preferably pMVDmaeB, pMV19maeB, or pMEmaeB, but is not limited thereto. Additionally, the host cell is a succinic acid-producing microorganism, a lactic acid-producing microorganism, or an ethanol-producing microorganism. The succinic acid-producing microorganism is the genus  Mannheimia  microorganism, and preferably, the genus  Mannheimia  microorganism in which one or more pathways selected from the group consisting of an acetate-producing pathway, a lactate-producing pathway, a formate-producing pathway, an ethanol-producing pathway and an oxaloacetate-producing pathway, were blocked. More preferably, the succinic acid-producing microorganism is a  Mannheimia  sp. LPK (KCTC 10558BP) or LPK7.  
         [0019]     As shown in a succinate synthesis pathway described more fully hereinafter with reference to  FIG. 1 , the maeB gene can convert malate to pyruvate. Thus, when the maeB gene is suitably expressed, it is possible to minimize malic acid that is produced as a byproduct in the production of succinic acid.  
         [0020]     Accordingly, the present invention relates in another aspect to a method for preparing succinic acid, the method including the steps of: culturing the recombinant microorganism; and recovering succinic acid from the culture broth of the recombinant microorganism. The steps of culturing the recombinant microorganism and recovering the succinic acid can be carried out by the culture method and the isolation and purification method of succinic acid, which are generally known in the prior fermentation industry.  
         [0021]     Additionally, by an increase in the production of pyruvate by the overexpression of the maeB gene, it is possible to increase the production of acetic acid, lactic acid or ethanol and to minimize the production of byproducts (succinic acid, malic acid and formic acid) in the preparation of acetic acid, lactic acid or ethanol. Thus, the present invention provides a method for preparing acetic acid, lactic acid or ethanol, the method including the steps of: culturing a microorganism producing acetic acid, lactic acid or ethanol, which is transformed with the maeB gene; and recovering lactic acid or ethanol from the microbial culture broth.  
         [0022]     Other aspects, features and embodiments of the present invention will be more fully apparent from the following detailed description and appended claims.  
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0023]      FIG. 1  is a schematic diagram showing a pathway for the synthesis of succinic acid from  Mannheimia  strain.  
         [0024]      FIG. 2  is a gene map of recombinant plasmid pMEmaeB.  
         [0025]      FIG. 3  is an SDS-PAGE showing the protein expression of recombinant  Mannheimia  LPK7pMEmaeB containing recombinant plasmid pMEmaeB. 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT  
       [0026]     The present invention is based on the discovery of a bacterial system for high productivity, low byproduct succinic acid production that overcomes the deficiencies of the prior art.  
         [0027]     The present inventors have made extensive efforts to find the core gene involved in succinic acid metabolism in order to develop a microbial strain capable of minimizing the production of malate and of producing succinic acid with higher yield, on the basis of a succinic acid synthetic pathway shown in  FIG. 1 , and as a result, they have cloned a malic enzyme B-encoding gene (maeB) derived from  Mannheimia succiniciproducens  MBEL55E and determined the function thereof, thereby completing the present invention.  
         [0028]     The present invention is more fully described hereinafter and with reference to illustrative examples. It is to be understood, however, that these examples are presented in order to more fully describe the present invention, and are correspondingly not intended to be construed to limit the present invention.  
         [0029]     Although only the use of the specified expression vector and the genus  Mannheimia  microorganism which is a succinic acid-producing microorganism, as a host cell, to express the inventive gene, is illustrated in the following examples, the use of other kinds of expression vectors and succinic acid-producing microorganisms will be obvious to a person skilled in the art. Also, it will be readily apparent to a person skilled in the art that the known acetic acid-producing microorganism, lactic acid-producing microorganism and ethanol-producing microorganism in place of the succinic acid-producing microorganism can be used as a host cell.  
       EXAMPLE 1  
     Preparation of  Mannheimia/E. coli  Shuttle Vector pME  
       [0030]      Mannheimia/E. coli  shuttle vector pME was prepared from pMVSCS1 reported to be isolated from  Mannheimia  (Kehrenberg et al.,  J. Antimicrob. Chemother.,  49:383, 2002) and  E. coli  expression vector pKK223-3 (Amersham Pharmacia Biotech). For this purpose, pKK223-3 was partially digested with BamHI and AccI to collect a 2.7kb fragment containing pBR322 ori and an ampicillin-resistant gene, and the single strand portions are filled with T4 DNA polymerase to make blunt ends. The blunt ends are ligated to prepare pKKD (2.7kb). pMVSCS1 (5.6kb) was digested with XhoII, and ligated with pKKD digested with restriction enzyme BamHI, to prepare fusion vector pMVD (8.3kb). The pMVD was digested with NcoI, and a 5.9kb fragment was religated to construct  Mannheimia/E. coli  shuttle vector pME.  
       EXAMPLE 2  
     Identification of Novel Gene (maeB) Derived From  Mannheimia Succiniciproducens  MBEL55E And Preparation of A Recombinant Plasmid Introduced With maeB Gene  
       [0031]     A malic enzyme-encoding gene of SEQ ID NO: 3 derived from  Mannheimia succiniciproducens  MBEL55E (KCTC 0769BP) was cloned, including a promoter and a transcription termination sequence.  
         [0032]     For this purpose, the chromosome of  Mannheimia succiniciproducens  MBEL55E as a template was subjected to PCR with primers of SEQ ID NOs: 1 and 2, under conditions shown in Table 1 below. The resulting maeB gene was digested with restriction enzyme BamHI and ligated to  Mannheimia/E. coli  shuttle vector pME digested with the same restriction enzyme to construct plasmid pMEmaeB ( FIG. 2 ). In this way, a malic enzyme-encoding gene (maeB) derived from  Mannheimia succiniciproducens  MBEL55E was cloned.  
                                                       TABLE 1                           Conditions for amplification of maeB gene.                    Restriction                   enzyme site               contained in       Gene   Primer   the primer   Reaction condition                    maeB   maeB-F(SEQ ID NO: 1),   BamH□   Cycle □:   94□, 5 min           maeB-R(SEQ ID NO: 2)       Cycle □:   (30 cycles)                       94□, 40 sec                       62□, 30 sec                       72□, 3 min                   Cycle □:   72□, 5 min                   Cycle □:   4□, forever                  
 
         [0033]     The DNA sequence of the cloned maeB of  Mannheimia succiniciproducens  MBEL55E was analyzed and the amino acid sequence of malic enzyme B was presumed. As a result, the maeB gene of  Mannheimia succiniciproducens  MBEL55E had a DNA sequence of 2,271 bp (SEQ ID NO: 3), and the malic enzyme B consisted of 757 amino acid residues (SEQ ID NO: 4).  
         [0034]     The homology of the maeB DNA sequence derived from  Mannheimia succiniciproducens  MBEL55E was analyzed, and as a result, this gene showed the highest homology of 82% (score: 196) with the malic enzyme of  Haemophilus ducreyi  35000HP, a homology of 80% (score: 194) with the malic enzyme of  Haemophilus influenza  Rd KW20, and a homology of 81% (score: 145) with the malic enzyme of  Escherichia coli  CFT073. Although there were studies on an NAD-dependent  E. coli -derived sfcA which is a malic enzyme gene, and the utilization thereof (Phue et al.,  Biotechnol. Bioeng.,  90:805, 2005),  Mannheimia succiniciproducens  MBEL55E has no sfcA gene and contains a NADP-dependent malic enzyme B-encoding gene maeB with the function similar to the sfcA gene.  
         [0035]     It was reported that  Actinobacillus  sp. known to produce a large amount of succinic acid together with  Mannheimia  showed about 6-8 times reduction in NADP-dependent malic enzyme activity when it was fermented with the addition of fumarate which is a product of malic enzyme reaction (Van der Werf et al.,  Arch. Microbiol.,  167:332, 1997). However, there are still no genome sequence of malic enzyme B and example showing the cloning and expression of the malic enzyme B.  
         [0036]     The G+C amount of the  Mannheimia succiniciproducens  MBEL55E maeB was found to be 46.2% which is higher than 38.5% for a  Haemophilus influenza  86-028NP maeA gene but lower than 54.3% for an  Escherichia coli  CFT073 maeB gene. Meanwhile, the frequency of using amino acid codons in the  Mannheimia succiniciproducens  MBEL55E maeB gene was examined and the results are shown in Table 2 below. As shown in Table 2 below, the frequency of using amino acid codons in the maeB gene showed a different result from that in generally known  E. coli.  For example, for the frequency of using lysine codons, AAA was used at a high frequency of 98% in the  Mannheimia succiniciproducens  MBEL55E maeB gene, but AAA and AAG were used at frequencies of 76% and 24%, respectively in generally known  E. coil.  For the frequency of using glutamate codons, GAA was used at a frequency of 94% in the  Mannheimia succiniciproducens  MBEL55E maeB gene, but GAA and GAG in  E. coli  were used at frequencies of 70% and 30%, respectively. Also, for the frequency of using glutamine codons, CAA and CAG were used at frequencies of 79% and 21%, respectively in the  Mannheimia succiniciproducens  MBEL55E maeB gene, but at frequencies of 31% and 69%, respectively in  E. coli.   
                                 TABLE 2                           Frequency of using amino acid codons                    Frequency of use   Average frequency       Amino acid   Codon   in MBEL55E maeB   of use in  E. coli                 Ala   GCA   0.20   0.22           GCC   0.24   0.25           GCG   0.43   0.34           GCT   0.14   0.19       Arg   AGA   —   0.04           AGG   —   0.03           CGA   —   0.05           CGC   0.55   0.37           CGG   —   0.08           CGT   0.45   0.42       Asn   AAC   0.30   0.61           AAT   0.70   0.39           GAC   —   0.41           GAT   —   0.59       Cys   TGC   0.56   0.57           TGT   0.44   0.43       STOP   TAA   —   0.62           TAG   —   0.09           TGA   —   0.30       Gln   CAA   0.79   0.31           CAG   0.21   0.69       Glu   GAA   0.94   0.70           GAG   0.06   0.30       Gly   GGA   0.14   0.09           GGC   0.35   0.40           GGG   0.06   0.13           GGT   0.45   0.38       His   CAC   0.56   0.48           CAT   0.44   0.52       Ile   ATA   0.02   0.07           ATC   0.38   0.46           ATT   0.61   0.47       Leu   CTA   0.04   0.03           CTC   0.12   0.10           CTG   0.12   0.55           CTT   0.11   0.10           TTA   0.45   0.11           TTG   0.17   0.11       Lys   AAA   0.98   0.76           AAG   0.02   0.24       Met   ATG   1.00   1.00       Phe   TTC   0.63   0.49           TTT   0.38   0.51       Pro   CCA   0.05   0.20           CCC   0.13   0.10           CCG   0.63   0.55           CCT   0.20   0.16       Ser   AGC   0.17   0.27           AGT   0.08   0.13           TCA   0.14   0.12           TCC   0.22   0.17           TCG   0.17   0.13           TCT   0.22   0.19       Thr   ACA   —   0.12           ACC   —   0.43           ACG   —   0.23           ACT   —   0.21       Trp   TGG   1.00   1.00       Tyr   TAC   0.40   0.47           TAT   0.60   0.53       Val   GTA   0.22   0.17           GTC   0.14   0.20           GTG   0.29   0.34           GTT   0.34   0.29                  
 
       EXAMPLE 3  
     Production of Succinic Acid By Use of Transformed  Mannheimia    
       [0037]     The recombinant plasmid pMEmaeB constructed in Example 2 was transformed into  Mannheimia  LPK7 (KCTC 10626BP) by electroporation to prepare LPK7pMEmaeB. Also, pME was introduced into  Mannheimia  LPK7 (KCTC 10626BP) to prepare LPK7pME.  
         [0038]     Each of the prepared recombinant strains was inoculated in 10 ml of a complex medium containing 9 g/l of glucose and cultured in an anaerobic condition at 39° C. for 16 hours. Each of the cultured strains was transferred in 250 ml of a complex medium containing 9 g/l of glucose and further cultured in the medium at 39° C. At this time, 100 μg/l of ampicillin as an antibiotic was added. The fermentation of each of the strains was performed by inoculating 250 ml of the  Mannheimia  culture broth in 2.5 L of a complex medium, and the fermentation conditions were as follows: initial glucose concentration: 20 g/l, pH: 6.8, and culture temperature: 39° C. For the adjustment of pH during the fermentation, ammonia solution (28%, v/v) was used, and the concentration of antibiotic ampicillin was the same as described above. A sample from each of the recombinant  Mannheimia  strains was collected during the fermentation, and the collected sample was centrifuged at 13,000 rpm and 4° C. for 10 minutes, and the concentrations of metabolites and succinic acid in the supernatant were analyzed by high-performance liquid chromatography (HPLC). The results are shown in Table 3 below.  
         [0039]     As shown in Table 3, in the case where the recombinant plasmid pMEmaeB containing the maeB gene of MBEL55E was introduced into the recombinant  Mannheimia  LPK7 (accession number: KCTC 10626BP), the concentration of malate was reduced but the concentration of pyruvate was increased. These results suggest that the maeB gene of MBEL55E encodes an enzyme involved in the mutual conversion between malate and pyruvate during several steps of the succinic acid-producing pathway. The reduction rate of malate in LPK7 was 137% which is much higher than that in the control group.  
                                                 TABLE 3                           Concentrations of malate and pyruvate in fermentation of transformed  Mannheimia                          Cell   Malate   Pyruvic acid   Malate   Succinic acid               Fermentation   concentration   concentration   concentration   reduction   concentration       Strain   Plasmid   time (hrs.)   (OD 600 )   (g/l)   (g/l)   rate (%)   (g/l)               LPK7   pME   25   3.08   2.58   2.40   100   12.98       LPK7   pMEmaeB   28   2.98   1.62   2.73   137   12.95                  
 
         [0040]     Meanwhile, each of the strains was analyzed by SDS-PAGE, and the results are shown in  FIG. 3 . As can be seen in  FIG. 3 , the recombinant  Mannheimia  LPK7pMEmaeB transformed with the recombinant plasmid pMEmaeB showed a remarkable increase in the expression of malic enzyme B as compared to the recombinant  Mannheimia  LPKpME (control group) transformed with pME.  
       EXAMPLE 4  
     Measurement of Malic Enzyme Activity By Use of Transformed  Mannheimia    
       [0041]     The culture broth of  Mannheimia  LPK7pMEmaeB prepared in Example 3 was centrifuged at 13,000 rpm and 4° C. for 5 minutes. The precipitated cells were washed 2 times with an iced buffer solution (100 mM Tris-HCI (pH 7.0), 20 mM KCl, 5 mM MnSO 4 , 2 mM DTT, 0.1 mM EDTA), and the washed cells were suspended in the same buffer and the cell membranes were disrupted by sonication. The cell debris were removed by a centrifugation, and the cell extract supernatant was used for the measurement of enzyme activity.  
         [0042]     The enzyme activity of the cell extracts was measured with a spectrophotometer, in which the cell extract was allowed to react by adding a reaction buffer (0.1 M Tris-HCl (pH 7.8), 5 mM MgCl 2 , 0.6 mM NADP+, 40 mM malate) to a 1 cm-width cuvette and adding the cell extract to the reaction buffer to a final volume of 1 ml, and the NADPH at 340 nm was measured. The results are shown in Table 4.  
         [0043]     As shown in Table 4, the LPK7pMEmaeB cell extract showed 120% increase in enzyme activity compared to the LPK7pME cell extract. This result confirms that the maeB gene according to the present invention is a gene encoding malic enzyme B having the activity of either converting pyruvate to malate or converting malate to pyruvate.  
                                 TABLE 4                           Enzyme activity of transformed  Mannheimia  strains                    *Enzyme   Enzyme activity       Strain   Plasmid   activity (U)   increase (%)               LPK7   pME   20.8   100       LPK7   pMEmaeB   25.1   120                 *Enzyme activity shows the titer of malic enzyme contained in 1 mg of total protein. An enzyme activity of 1.0 U is defined as the amount of enzyme required for converting 1 nmole of a substrate to a certain product at 37° C. for 1 minute.             
 
         [0044]     The activity of the malic enzyme according to the present invention was compared to the known enzyme, and the result is shown in Table 5 below. As shown in Table 5, the malic enzyme of the  Mannheimia  strain transformed with the inventive maeB gene showed much higher activity than the malic enzyme of  E. coli  K12 (Gray et al.,  Biochim. Biophys. Acta,  117:33, 1966).  
                                                   TABLE 5                           Comparison of malic enzyme activities between       transformed  Mannheimia  and  E. coli                      Enzyme   Gene           Strain   activity (U)   homology (%)                            LPK7pMEmaeB   25.1   67.6             E. coli  K12   &lt;1                      
 
         [0045]     As described and proven in detail above, the present invention provides a novel gene encoding the malic enzyme B. The nucleotide sequence of the maeB gene may be a DNA sequence of SEQ ID NO: 3, or a sequence having appropriate homology thereto (e.g., that is at least 85%, and more preferably is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homologous to the nucleotide sequence of SEQ ID NO: 3). The novel gene according to the present invention is useful to prepare a recombinant microorganism capable of effectively reducing malate produced as a byproduct in the production of succinic acid. Also, the expression of the maeB gene can lead to an increase in the production of pyruvate during the preparation of acetic acid, lactic acid or ethanol. Thus, the maeB gene can increase the production of acetic acid, lactic acid or ethanol, and is also useful to prepare a microorganism for minimizing the production of byproducts (succinate, malate and formate). Accordingly, the maeB gene according to the present invention will be useful in increasing the production of various metabolites in the operation of central metabolic pathways by the combination with a suitable metabolic pathway.  
         [0046]     While the present invention has been described in detail with reference to specific features, it will be apparent to those skilled in the art that this description is illustrative only of a preferred embodiment and is not intended in any way to limit the scope of the present invention, as defined by the appended claims and equivalents thereof.