MICROORGANISM HAVING ABILITY TO PRODUCE O-SUCCINYLHOMOSERINE OR SUCCINIC ACID, AND METHOD FOR PRODUCING SUCCINIC ACID OR O-SUCCINYLHOMOSERINE BY USING SAME

Abstract

Provided are a microorganism having an ability to produce O-succinylhomoserine or succinic acid, and a method of producing O-succinylhomoserine or succinic acid by using the same.

Claims

1. A microorganism of the genus Escherichia having an ability to produce O-succinylhomoserine or succinic acid, wherein an activity of α-ketoglutarate dehydrogenase complex is enhanced compared to an endogenous activity level thereof.

2. The microorganism according to claim 1, wherein the α-ketoglutarate dehydrogenase complex comprises amino acid sequences set forth in SEQ ID NO: 22 and SEQ ID NO: 24.

3. The microorganism according to claim 1, wherein an activity of homoserine O-succinyltransferase is further enhanced compared to an endogenous activity level thereof.

4. The microorganism according to claim 1, wherein activity of at least one of cystathionine gamma-synthase and homoserine kinase, is further weakened compared to corresponding endogenous activity levels, or eliminated.

5. The microorganism according to claim 1, wherein the microorganism is Escherichia coli.

6. A method of producing O-succinylhomoserine or succinic acid, comprising: culturing a microorganism of the genus Escherichia having an ability to produce O-succinylhomoserine or succinic acid in a media, wherein an activity of α-ketoglutarate dehydrogenase complex is enhanced compared to an endogenous activity level thereof; and recovering O-succinylhomoserine or succinic acid from the culture media or the cultured microorganism.

7. The method according to claim 6, wherein the microorganism is Escherichia coli.

8. The microorganism according to claim 2, wherein the microorganism is Escherichia coli.

9. The microorganism according to claim 3, wherein the microorganism is Escherichia coli.

10. The microorganism according to claim 4, wherein the microorganism is Escherichia coli.

Description

DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1 is a schematic diagram of the recombinant vector pCL_PsucA-sucAB.

[0031] FIG. 2 is a schematic diagram of the recombinant vector pCL_Prmf-sucAB.

[0032] FIG. 3 is a schematic diagram of the recombinant vector pCL_Ptrc-sucAB.

[0033] FIG. 4 is a schematic diagram of the recombinant vector pCL_Pcysk-metA11_PsucA-sucAB.

MODE OF THE INVENTIVE CONCEPT

[0034] Hereinafter, the present disclosure will be described in more detail with reference to Examples. However, these Examples are for illustrative purposes only, and the scope of the present disclosure is not intended to be limited by these Examples.

Reference Example. Construction of O-Succinylhomoserine- or Succinic Acid-Producing Strain

[0035] 1) Deletion of metB Gene

[0036] To increase the accumulation of O-succinylhomoserine or succinic acid, a strain was constructed by deleting metB gene encoding cystathionine gamma-synthase involved in the degradation of L-methionine precursor.

[0037] The metB gene encoding cystathionine gamma-synthase in the wild-type E. coli (K12) W3110 strain was deleted. It is known that Cystathionine gamma-synthase binds to various methionine precursors and thereby can produce various by-products. Thus, the overexpression of cystathionine synthase might increase side reactions to reduce efficiency of intracellular reactions. To delete the metB gene, FRT-one-step-PCR deletion method was performed (PNAS (2000), Vol. 197, p 6640-6645). To delete the metB gene, PCR was performed with the primers represented by SEQ ID NO: 12 and SEQ ID NO: 13 using the pKD3 vector (PNAS (2000) Vol. 97, p 6640-6645) as a template, resulting in the construction of a deletion cassette.

TABLE-US-00001 SEQ ID NO: 12 5′-TTACTCTGGTGCCTGACATTTCACCGACAAAGCCCAGGGAACTTCA TCACGTGTAGGCTGGAGCTGCTTC-3′; SEQ ID NO: 13 5′-CGCTGCGCCAGCTCCATACGCGGCACCAGCGTTCGCAACCCACGT AGCAGCATATGAATATCCTCCTTAG-3′;

[0038] PCR was performed as follows; denaturation at 95□ for 30 seconds, annealing at 55□ for 30 seconds, and extension at 72□ for 1 minute, with 30 cycles performed from denaturation to extension. The PCR product underwent electrophoresis on 1.0% agarose gel. The 1.1 kb band was eluted and purified. The recovered DNA fragment was electroporated into the E. coli (K12) W3110 strain previously transformed with the pKD46 vector (PNAS (2000) Vol. 97, p 6640-6645). For the electroporation, the W3110 strain transformed with pKD46 was cultured in LB medium supplemented with 200 μg/L ampicillin and 5 mM L-arabinose at 30□ until OD.sub.600 reached 0.5. The strain was washed with 10% glycerol three times. Electroporation was performed at 2500 V. The recovered strain was spread on LB plate medium containing 30 μg/L chloramphenicol, followed by culturing at 37□ for 1 to 2 days. Then, the resistant strain was selected.

[0039] The selected strain was then used for PCR using the primers represented by SEQ ID NO: 14 and SEQ ID NO: 15 according to the conditions mentioned above.

[0040] A band of a size of 1.5 kb was observed on 1.0% agarose gel, suggesting that the metB gene was deleted.

TABLE-US-00002 SEQ ID NO: 14 5′-TATTCGCCGCTCCATTCAGC-3′; SEQ ID NO: 15 5′-TACCCCTTGTTTGCAGCCCG-3′;

[0041] The metB gene-deleted strain was transformed with the pCP20 vector (PNAS (2000) vol. 97, p 6640-6645), which was then cultured in LB medium supplemented with 100 μg/L ampicillin. PCR was performed by the same manner as described above and the final metB gene-deleted strain, exhibiting a shrunk band on 1.0% agarose gel, was selected. The deletion of chloramphenicol marker was confirmed. The obtained methionine-auxotrophic strain was named CC03-0132.

[0042] 2) Deletion of thrB Gene

[0043] To increase the production of O-succinyl homoserine from homoserine, thrB, the gene encoding homoserine kinase, was deleted. In particular, when a threonine-producing strain is used, the deletion of this gene is necessary because homoserine utilization activity is very strong. To delete the thrB gene from the CC03-0132 strain constructed in 1) above, FRT-one-step-PCR deletion method (PNAS (2000) Vol. 97, p 6640-6645) was performed. To delete the thrB gene, PCR was performed with the primers represented by SEQ ID NO: 16 and SEQ ID NO: 17 using the pKD3 vector (PNAS (2000) Vol. 97, p 6640-6645) as a template, resulting in the construction of a deletion cassette.

TABLE-US-00003 SEQ ID NO: 16 5′-CATGGTTAAAGTTTATGCCCCGGCTTCCAGTGCCAATATGAGCGTC GGGTGTGTAGGCTGGAGCTGCTTC-3′; SEQ ID NO: 17 5′-GGAGATACCGCTCGCTACCGCGCCGATTTCCGCGACCGCCTGCC GCGCCTCATATGAATATCCTCCTTAG-3′;

[0044] PCR was performed as follows; denaturation at 95□ for 30 seconds, annealing at 55□ for 30 seconds, and extension at 72□ for 1 minute, with 30 cycles performed from denaturation to extension. The PCR product underwent electrophoresis on 1.0% agarose gel. The 1.1 kb band was eluted and purified. The recovered DNA fragment was electroporated into the CC03-0132 strain previously transformed with the pKD46 vector (PNAS (2000) Vol. 97, p 6640-6645). For the electroporation, the CC03-0132 strain transformed with pKD46 was cultured in LB medium supplemented with 200 μg/L ampicillin and 5 mM L-arabinose at 30□ until OD.sub.600 reached 0.5. The strain was washed with 10% glycerol three times. Electroporation was performed at 2500 V. The recovered strain was spread on LB plate medium containing 30 μg/L chloramphenicol, followed by culturing at 37□ for 1 to 2 days. Then, the resistant strain was selected.

[0045] The selected strain proceeded to PCR using the primers represented by SEQ ID NO: 18 and SEQ ID NO: 19 according to the conditions mentioned above. A band of a size of 1.5 kb was observed on 1.0% agarose gel, suggesting that the thrB gene was deleted.

TABLE-US-00004 SEQ ID NO: 18 5′-ACTCGACGATCTCTTTGCC-3′; SEQ ID NO: 19 5′-ACGCCGAGAGGATCTTCGCAG-3′;

[0046] The confirmed strain was transformed with the pCP20 vector (PNAS (2000) vol. 97, p 6640-6645), which was then cultured in LB medium supplemented with 100 μg/L ampicillin. PCR was performed thereon in the same manner as described above and the final thrB gene-deleted strain, exhibiting a smaller band as confirmed on 1.0% agarose gel electrophoresis, was selected. The deletion of the chloramphenicol marker was confirmed. The obtained strain was named CC03-0133.

[0047] 3) Construction of pSG Vector for Inserting metA11

[0048] Homoserine succinyl transferase is under feedback control by trace methionine added to the medium so that most of the activity of homoserine succinyl transferase is inhibited. To increase the production of O-succinyl homoserine, the L-methionine precursor, a mutant free from feedback control by methionine, was used. To replace the wild-type metA gene (SEQ ID NO: 27) on the chromosome encoding homoserine succinyl transferase in E. coli with the metA11 (SEQ ID NO: 33) mutant which is free from feedback control by methionine, the insertion vector pSG-metA11 was constructed. According to the instruction given in WO2008/127240 A1, the nucleotide sequence information of the metA11 gene was obtained, and based on the information, the primers (SEQ ID NO: 20 and SEQ. ID NO: 21) were synthesized containing the open reading frame (ORF) starting from the start codon ATG of the metA11 gene and the recognition sites of restriction enzymes, EcoRI and SacI. PCR was performed with the primers represented by the following sequences using TF4076BJF metA#11 strain shown in WO2008/127240 A1 as a template.

TABLE-US-00005 SEQ ID NO: 20 5′-ggccgaattcatgccgattcgtgtgccgga-3′; SEQ ID NO: 21 5′-ggccgagttcttaatccagcgttggattca-3′;

[0049] PCR was performed using pfu-X DNA polymerase (Solgent, SPX16-R250) as follows: denaturation at 95□ for 30 seconds, annealing at 55□ for 30 seconds, and extension at 72 □ for 2 minutes, with 30 cycles from denaturation to extension. As a result, the PCR product in which the metA11 ORF containing the recognition sites of EcoRI and SacI at both ends was amplified, was obtained. The metA11 gene obtained by PCR above was treated with the restriction enzymes EcoRI and SacI, followed by ligation into the pSG76-C vector (J Bacteriol. 1997 July; 179(13):4426-8.) which had also been treated with the restriction enzymes EcoRI and SacI. Finally, the pSG-metA11 recombinant vector containing the cloned metA11 gene was constructed.

[0050] 4) Construction of metA11 Insertion Strain

[0051] The metA11 insertion vector pSG-metA11 constructed in Reference Example 3) was introduced into the strain obtained in Reference Example 2), followed by culture in LB-Cm medium (yeast extract 10 g/L, NaCl 5 g/L, tryptone 10 g/L, chloramphenicol 30 μg/L). Then, chloramphenicol-resistant colonies were selected. The selected transformant was the primary strain inserted with the pSG-metA11 vector in the position of metA of the chromosome. The strain introduced with the metA11 gene was transformed with the pASceP vector (JOURNAL OF BACTERIOLOGY, July 1997, 4426-4428) expressing the restriction enzyme I-Scel to cleave the I-Scel site in the pSG vector. The strain was selected after being grown in LB-Amp medium (yeast extract 10 g/L, NaCl 5 g/L, tryptone 10 g/L, chloramphenicol 100 μg/L). In the selected strain, the wild-type metA was replaced with metA11 and the inserted pSG76-C vector was eliminated therefrom. This strain was named E. coli CC03-0038.

[0052] 5) Construction of O-Succinylhomoserine-Producing Strain Based on Threonine-Producing Strain

[0053] A strain having an ability to produce succinic acid and O-succinyl homoserine was constructed by the same method as described in 1) to 4) above by using E. coli KCCM 10541P, the threonine production strain, described in International Patent WO 2005/075625, instead of the wild-type strain W3110, and the constructed strain was named CJM2-A11.

Example 1. Construction of a Plasmid for the Enhanced Expression of sucAB

[0054] 1-1. Construction of a Plasmid for the Enhanced Expression of sucAB with a sucA Promoter

[0055] The nucleotide sequence information of α-ketoglutarate dehydrogenase encoded by sucAB (sucA GenBank Accession No. BAA35392.1: SEQ ID NO: 23, sucB GenBank Accession No. BAA35393.1: SEQ ID NO: 25) was obtained from the database of the National Center for Biotechnology Information, USA, based on which the primers, represented by SEQ ID NO: 1 and SEQ ID NO: 2, and recognized by the restriction enzymes HindIII and XbaI respectively and containing the sequence ranging −188 from ATG, the ORF initiation codon of sucAB, were synthesized in order to obtain the sucAB gene under the control of the sucA promoter.

TABLE-US-00006 SEQ ID NO: 1 5′-GGCCAAGCTTGCATCAGGCGTAACAAAGAA-3′; SEQ ID NO: 2 5′-GGCCTCTAGATGTCCATCCTTCAGTAATCG-3′;

[0056] Using the chromosomal DNA of wild-type E. coli W3110 as a template, the cloning of sucAB, the gene encoding α-ketoglutarate dehydrogenase, was performed by PCR with the primers represented by SEQ ID NO: 1 and SEQ ID NO: 2. PCR [Sambrook et al, Molecular Cloning, a Laboratory Manual (1989), Cold Spring Harbor Laboratories] was performed using pfu-X DNA polymerase (Solgent, SPX16-R250) as follows: denaturation at 95□ for 30 seconds, annealing at 55□ for 30 seconds, and extension at 72□ for 5 minutes, with 30 cycles from denaturation to extension. As a result, an approximately 4.5 kb PCR product containing the sucA promoter, the sucAB gene, and the recognition sites of HindIII and XbaI was obtained. The obtained PCR product was treated with the restriction enzymes HindIII and XbaI. By using T4 DNA ligase, the PCR product was ligated into the pCL1920 vector (Lerner, C. G. and Inouye, M., Nucl. Acids Res. (1990) 18:4631) which had been pre-treated with the restriction enzymes HindIII and XbaI, resulting in the construction of the recombinant vector pCL PsucA-sucAB. FIG. 1 is a schematic diagram illustrating the recombinant vector pCL PsucA-sucAB.

[0057] 1-2. Construction of a Plasmid for the Enhanced Expression of sucAB with a Promoter rmf or trc

[0058] The nucleotide sequence information (sucA GenBank Accession No. BAA35392.1: SEQ ID NO: 23, sucB GenBank Accession No. BAA35393.1: SEQ ID NO: 25) of the sucAB gene (the gene encoding α-ketoglutarate dehydrogenase) was obtained from the database of the National Center for Biotechnology Information, USA, based on which the primers represented by SEQ ID NO: 3 and SEQ ID NO: 4 having the recognition sites of EcoRV and HindIII were synthesized in order to obtain the sucAB gene.

TABLE-US-00007 SEQ ID NO: 3 5′-ATCATGCAGAACAGCGCTTTGAA-3′; SEQ ID NO: 4 5′-GGCCAAGCTTTGTCCATCCTTCAGTAATCG-3′;

[0059] Using the chromosomal DNA of wild-type E. coli W3110 as a template, the cloning of sucAB was performed by PCR with the primers represented by SEQ ID NO: 3 and SEQ ID NO: 4. FOR [Sambrook et al, Molecular Cloning, a Laboratory Manual (1989), Cold Spring Harbor Laboratories] was performed using pfu-X DNA polymerase (Solgent, SPX16-R250) as follows: denaturation at 95□ for 30 seconds, annealing at 55 □ for 30 seconds, and extension at 72 □ for 5 minutes, with 30 cycles from denaturation to extension. As a result, an approximately 4.3 kb PCR product containing the sucAB gene and the recognition site of restriction enzyme HindIII was obtained. The obtained PCR product was treated with the restriction enzyme HindIII. To replace the sucA promoter, that is, the native promoter of the sucAB gene, the vectors pCL_Prmf-gfp (SEQ ID NO: 5) and pCL_Ptrc-gfp (SEQ ID NO: 6) containing respectively promoters rmf and trc were treated with the restriction enzymes EcoRV and HindIII, followed by ligation of the PCR product using T4 DNA ligase (Roche: 10481220001). As a result, the recombinant vectors pCL_Prmf-sucAB and pCL_Ptrc-sucAB were constructed. The vectors pCL_Prmf-gfp and pCL_Ptrc-gfp were introduced with the green fluorescence protein gene gfp in order to measure the strength of the promoters rmf and trc. In the meantime, sucAB was ligated with the promoter region of the vector, resulting in the construction of a vector containing sucAB that would be expressed under the control of the promoters rmf and trc. FIG. 2 and FIG. 3 are schematic diagrams illustrating the recombinant vectors pCL_Prmf-sucAB and pCL_Ptrc-sucAB, respectively.

Example 2. Construction of a Plasmid for the Simultaneously Enhanced Expression of sucAB and metA11

[0060] To synthesize O-succinylhomoserine, an expression vector expressing sucAB and metA11 simultaneously was constructed. The nucleotide sequence information of the metA11 gene was obtained based on the amino acid sequence encoding the O-succinyl transferase mutant of the TF4076BJF metA#11 strain described in International Patent Publication No. WO2008/127240 A1, and, based on the nucleotide sequence information, the primers represented by SEQ ID NO: 7 and SEQ ID NO: 8 having the recognition sites of restriction enzymes EcoRV and HindIII at both ends were synthesized in order to amplify the ORF ranging from ATG to TAA of the metA11 gene.

TABLE-US-00008 SEQ ID NO: 7 5′-GAGTGCGATATC atgccgattcgtgtgccggac-3′; SEQ ID NO: 8 5′-GCACTCAAGCTT ttaatccagcgttggatacatg-3′;

[0061] Using TF4076BJF metA#11 as a template, PCR was performed with the primers represented by SEQ ID NO: 7 and SEQ ID NO: 8. PCR was performed using pfu-X DNA polymerase (Solgent, SPX16-R250) as follows: denaturation at 95□ for 30 seconds, annealing at 55□ for 30 seconds, and extension at 72□ for 1 minute, with 30 cycles from denaturation to extension. The obtained PCR product was treated with the restriction enzymes EcoRV and HindIII. The vector pCL_Pcysk-gfp (SEQ ID NO: 9) was treated with the restriction enzymes EcoRV and HindIII, followed by ligation using T4 DNA ligase (Roche: 10481220001). As a result, the recombinant vector pCL_Pcysk-metA11 was constructed. To insert sucAB in the constructed vector above, the primers represented by SEQ ID NO: 10 and SEQ ID NO: 11 having the recognition site of HindIII were synthesized.

TABLE-US-00009 SEQ ID NO: 10 5′-GGCCAAGCTTGCATCAGGCGTAACAAAGAA-3′; SEQ ID NO: 11 5′-GGCCAAGCTTTGTCCATCCTTCAGTAATCG-3′;

[0062] To express metA11 and sucAB simultaneously, the vector pCL_Pcysk-metA11_PsucA-sucAB was constructed. First, using the chromosomal DNA of wild-type E. coli W3110 as a template, PCR was performed with the primers represented by SEQ ID NO: 10 and SEQ ID NO: 11. FOR [Sambrook et al, Molecular Cloning, a Laboratory Manual (1989), Cold Spring Harbor Laboratories] was performed using pfu-X DNA polymerase (Solgent, SPX16-R250) as follows: denaturation at 95□ for 30 seconds, annealing at 55□ for 30 seconds, and extension at 72 □ for 5 minutes, with 30 cycles from denaturation to extension. As a result, a PCR product of approximately 4.5 kb having PsucA-sucAB including the recognition site of HindIII was obtained. The obtained PCR product was treated with the restriction enzyme HindIII, followed by ligation, using T4 DNA ligase (Roche:10481220001), into the pCL_Pcysk-metA11 vector which had been pre-treated with the restriction enzyme HindIII. As a result, the recombinant vector pCL_Pcysk-metA11_PsucA-sucAB was constructed. FIG. 4 is a schematic diagram illustrating the recombinant vector pCL_Pcysk-metA11_PsucA-sucAB.

Example 3. Fermentation for the Production of Succinic Acid

[0063] Flask culture was performed to investigate the production of succinic acid when activity of only sucAB was enhanced in the O-succinylhomoserine-producing strain constructed in the Reference Example. The strain of Reference Example 4), CC03-0038, and the strain of Reference Example 5), CJM2-A11, were transformed with the plasmids constructed in Example 1, pCL_PsucA-sucAB, pCL_Prmf-sucAB, and pCL_Ptrc-sucAB. The strains were spread on an LB plate medium containing spectinomycin, resulting in strains inserted with sucAB. As controls, prepared were CC03-0038 and CJM2-A11 strains introduced with the pCL1920 vector. The pCC1BAC-scrO vector (SEQ ID NO: 34) having the scrO sequence of the pUR 400 plasmid originating from the salmonella strain described in International Patent Publication No. WO10/101360 was introduced into these strains to make them to use raw sugar as a carbon source. The resultant strains were cultured in the medium having the composition shown in Table 2 below for the evaluation of the production of succinic acid.

[0064] Each strain was inoculated into the medium and cultured at 33□ overnight. A single colony was inoculated into 2 ml of LB medium containing spectinomycin, followed by culture at 33□ for 2 hours. The strain was inoculated again into a 250 ml Erlenmeyer flask containing 25 ml flask medium at a density of OD.sub.500=0.5, followed by culture at 33□ at 200 rpm for 33 hours. High Performance Liquid Chromatography (HPLC) was performed to investigate the production of succinic acid. The results are shown in Table 3.

[0065] As a result, as expression of sucAB was enhanced, the production of succinic acid was increased. This result indicates that the enhanced expression of sucAB has the effect of down-regulating glutamate but increasing the flux of succinyl-CoA production to increase the production of succinic acid. When glucose was used as a carbon source, the production of succinic acid increased by 30% at maximum. The production of succinic acid was expected to be further increased as succinic acid synthesis was enhanced in a strain under aerobic conditions. When raw sugar was used as a carbon source, the production of succinic acid increased as much as when glucose was used as a carbon source. The results are shown in Table 4.

TABLE-US-00010 TABLE 1 Composition of glucose flask medium Composition Stock Conc (per liter) Vol (ml) Glucose 40 g 200 KH.sub.2PO.sub.4 2 g 100 Ammonium sulfate 17 g 500 MgSO.sub.4•7H.sub.2O 1 g Yeast extract 2 g Methionine 0.4 g Threonine 1 g MnSO.sub.4•7H.sub.2O 10 mg/ml 0.01 g (stock 1 ml) ZnSO.sub.4•7H.sub.2O  1 mg/ml 0.01 g (stock 10 ml) FeSO.sub.4•7H.sub.2O 10 mg/ml 10 mg (stock 1 ml) Calcium carbonate 30 g 200

TABLE-US-00011 TABLE 2 Composition of raw sugar flask medium Composition Stock Conc (per liter) Vol (ml) Raw sugar 60 g 200 KH.sub.2PO.sub.4 2 g 100 Ammonium sulfate 25 g 500 MgSO.sub.4•7H.sub.2O 1 g Yeast extract 2 g methionine 0.4 g threonine 1 g MnSO.sub.4•7H.sub.2O 10 mg/ml 0.01 g (stock 1 ml) ZnSO.sub.4•7H.sub.2O  1 mg/ml 0.01 g (stock 10 ml) FeSO.sub.4•7H.sub.2O 10 mg/ml 10 mg (stock 1 ml) Calcium carbonate 30 g 200

TABLE-US-00012 TABLE 3 Succinic acid production via flask culture using glucose O- succinic O-succinyl Gluta- Glucose acid homoserine mate Strain (g/L) (g/L) (g/L) (g/L) CC03-0038/pCL1920 40 0.30 4.5 1.87 CC03-0038/ 40 0.63 4.9 0.0 pCL_PsucA-sucAB CC03-0038/ 40 0.88 5.2 0.0 pCL-Prmf-sucAB CC03-0038/pCL_Ptrc-sucAB 40 0.93 5.5 0.0 CJM2-A11/pCL1920 40 0.012 10.9 0.84 CJM2-A11/pCL_PsucA- 40 0.26 11.5 0.0 sucAB CJM2-A11/pCL-Prmf-sucAB 40 0.63 12.1 0.0 CJM2-A11/pCL_Ptrc-sucAB 40 0.77 12.5 0.0

TABLE-US-00013 TABLE 4 Succinic acid production via flask culture using raw sugar Raw O-succinyl Gluta- sugar O-succinic homoserine mate Strain (g/L) acid (g/L) (g/L) (g/L) CC03-0038/pCC1BAC-scrO/ 60 0.24 7.0 1.33 pCL1920 CC03-0038/pCC1BAC-scrO/ 60 0.312 7.5 0 pCL_PsucA-sucAB CC03-0038/pCC1BAC-scrO/ 60 0.762 7.9 0 pCL_Prmf-sucAB CC03-0038/pCC1BAC-scrO/ 60 1.56 8.2 0 pCL_Ptrc-sucAB CJM2-A11/pCC1BAC-scrO/ 60 0.023 15.7 0.77 pCL1920 CJM2-A11/pCC1BAC-scrO/ 60 0.22 16.1 0 pCL_PsucA-sucAB CJM2-A11/pCC1BAC-scrO/ 60 0.53 16.7 0 pCL-Prmf-sucAB CJM2-A11/pCC1BAC-scrO/ 60 1.07 17.0 0 pCL_Ptrc-sucAB

Example 4. Fermentation for the Production of O-Succinyl Homoserine

[0066] To enhance the expression of sucAB and metA genes together in the CC03-0038 and CJM2-A11 strains, the strains were transformed with the pCL_Pcysk-metA11_PsucA-sucAB plasmid constructed in Example 2, followed by culturing in an Erlenmeyer flask to investigate the production of O-succinyl homoserine. The composition of the flask medium was as shown in Table 1. The enhanced expression of metA11 gene in Reference Example 4) was under the control of the original promoter. Therefore, in order to enhance its expression, metA11 was cloned into a vector where it could be expressed by the cysK promoter, wherein the expression of sucAB was simultaneously induced.

[0067] CC03-0038 was transformed with the pCL_Pcysk-metA11_PsucA-sucAB plasmid constructed in Example 2. The strain was spread on the LB plate medium containing spectinomycin, resulting in the preparation of the transformed strain. As for the controls, CC03-0038 and CJM2-A11 were transformed with the pCL1920 vector. A strain that could use raw sugar as a carbon source was constructed by introducing the pCC1BAC-scrO vector having the scrO sequence of the pUR 400 plasmid originating from the salmonella strain described in Korean Patent Application No. 2009-0018128 into the strains. The strain was cultured in the raw sugar flask medium having the composition shown in Table 2 for the evaluation of the production of O-succinyl homoserine. For evaluation purpose, some of the strains described above were inoculated into the medium and cultured at 33□ overnight. A single colony was inoculated into 2 ml of LB medium containing spectinomycin, followed by culture at 33□ for 2 hours. The strains were inoculated again into a 250 ml Erlenmeyer flask containing 25 ml flask medium at a density of OD.sub.500=0.5, followed by culture at 33□ at 200 rpm for 33 hours. HPLC was performed to investigate the production of O-succinylhomoserine. The results are shown in Table 5.

[0068] The results indicated that, in a strain introduced with pCL_Pcysk-metA11_PsucA-sucAB, O-succinylhomoserine was up-regulated. The production yield was increased by about 40% as compared with the control. As O-succinylhomoserine was increased, glutamate and homoserine were down-regulated. The results indicate that the enhanced expression of sucAB plays an important role in supplying succinyl-CoA. When expression of metA, using succinyl-CoA as a substrate, was simultaneously enhanced, the concentration of O-succinylhomoserine rapidly increased.

TABLE-US-00014 TABLE 5 O-succinylhomoserine production via flask culture using glucose O-succinyl homoserine Glutamate Homoserine Strain (g/L) (g/L) (g/L) CC03-0038/pCL1920 4.5 1.87 0.4 CC03-0038/ 6.31 0.0 0.17 pCL_Pcysk-metA11_PsucA- sucAB CJM2-A11/pCL1920 10.9 0.84 1.1 CJM2-A11/pCL_Pcysk- 15.8 0.0 0.5 metA11_PsucA-sucAB

TABLE-US-00015 TABLE 6 O-succinylhomoserine production via flask culture using raw sugar O-succinyl homoserine Glutamate Homoserine Strain (g/L) (g/L) (g/L) CC03-0038/pCC1BAC-scrO/ 7.0 1.87 0.7 pCL1920 CC03-0038/ 12.4 0.0 0.23 pCC1BAC-scrO/ pCL_Pcysk- metA11_PsucA-sucAB CJM2-A11/pCC1BAC-scrO/ 15.7 0.84 1.5 pCL1920 CJM2-A11/pCC1BAC-scrO/ 22.6 0.0 0.54 pCL_Pcysk- metA11_Pna-sucAB

[0069] When a microorganism belonging to the genus Escherichia was transformed with a vector containing sucAB according to an embodiment of the present disclosure to enhance the expression of sucAB, the production of succinyl-CoA and succinic acid was increased. Therefore, the enhanced expression of sucAB may be applied to those microorganisms that have the same TCA cycle in regard to central carbon metabolism as the above, for example to those microorganisms such as yeast and Actinomyces, etc.

[0070] The engineered CC03-0038/pCL_Pcysk-metA11_PsucA-sucAB strain, confirmed to be capable of producing O-succinyl homoserine, was named CC03-0157, and was deposited at the Korean Culture Center of Microorganisms (KCCM) under the Budapest Treaty on Nov. 22, 2013 (Accession No: KCCM11488P).

Sequence Listing Free Text

[0071] The sequences represented by SEQ ID NO: 1 to SEQ ID NO: 34 described used herein are shown in the attached sequence list.