Microorganisms for production of O-succinylhomoserine and method for production of O-succinylhomoserine using the same

Abstract

The present invention relates to a polypeptide having a resistant to feedback inhibition by methionine and an activity of homoserine O-succinyltransferase, a O-succinyl homoserine-producing microorganism expressing the polypeptide, and a method for producing O-succinyl homoserine using the same.

Claims

1. An O-succinylhomoserine-producing Escherichia sp. microorganism expressing a polypeptide having a resistance to feedback inhibition by methionine and a homoserine succinyltransferase activity, wherein the polypeptide has the amino acid sequence of SEQ ID NO: 1.

2. A method of producing O-succinylhomoserine, comprising: (a) culturing the microorganism of claim 1 in a medium; and (b) obtaining O-succinylhomoserine from the microorganism or the medium.

3. The O-succinylhomoserine-producing Escherichia sp. microorganism of claim 1, wherein the Escherichia sp. microorganism is Escherichia coli.

4. A method of producing O-succinylhomoserine, comprising: (a) culturing the microorganism of claim 3 in a medium; and (b) obtaining O-succinylhomoserine from the microorganism or the medium.

5. The Escherichia sp. microorganism of claim 1, wherein the metB gene encoding cystathionine gamma synthase is further deleted or weakened.

6. A method of producing O-succinylhomoserine, comprising: (a) culturing the microorganism of claim 5 in a medium; and (b) obtaining O-succinylhomoserine from the microorganism or the medium.

7. The Escherichia sp. microorganism of claim 1, wherein the thrB gene encoding homoserine kinase or the metA gene encoding homoserine O-succinyltransferase are further deleted or weakened.

8. A method of producing O-succinylhomoserine, comprising: (a) culturing the microorganism of claim 7 in a medium; and (b) obtaining O-succinylhomoserine from the microorganism or the medium.

9. A method of producing methionine, comprising: (a) culturing the microorganism of claim 1 in a medium; (b) obtaining O-succinylhomoserine from the microorganism or the medium; (c) converting the O-succinylhomoserine into methionine by using cystathionine gamma synthase or O-succinylhomoserine sulfhydrylase.

10. The method of producing methionine of claim 9, wherein the Escherichia sp. microorganism is Escherichia coli.

11. The method of producing methionine of claim 9, wherein the metB gene encoding cystathionine gamma synthase of the Escherichia sp. microorganism is further deleted or weakened.

12. The method of producing methionine of claim 9, wherein the thrB gene encoding homoserine kinase or the metA gene encoding homoserine O-succinyltransferase of the Escherichia sp. microorganism are further deleted or weakened.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) Hereinafter, the present invention will be described in more detail with reference to the following Examples. However, these Examples are for illustrative purposes only, and the invention is not intended to be limited by these Examples.

EXAMPLE 1

Selection of Polypeptides Having Novel O-Succinyltransferase Activity

(2) As a method of releasing the feedback control of metA gene and securing the stability thereof, metX derived from Chromobacterium violaceum has been developed based on the fact that the metX gene (homoserine O-acetyltransferase) is not feedback inhibited by L-methonine although the metX gene has a structure similar to that of metA gene.

(3) In this regard, for the development of a novel homoserine O-acetyltransferase, the present inventors have performed a homology analysis regarding the amino acid sequences of the already-developed metX derived from Chromobacterium violaceum, and finally selected the polypeptide having an amino acid sequence of SEQ ID NO: 1, released from the feedback inhibition by methionine. The present inventors have newly confirmed that the selected polypeptide, although it is a metX derived from Sideroxydans lithotrophicus ES-1, has a novel activity that has never been reported previously.

EXAMPLE 2

Plasmid Construction

(4) 2-1. Synthesis of metX Gene Derived from Sideroxydans lithotrophicus ES-1

(5) The metX gene derived from Sideroxydans lithotrophicus ES-1 (sli) (SEQ ID NO: 3) was synthesized based on the metX gene sequence (SEQ ID NO: 2) of the NCBI database (reference sequence: YP_003522665.1) via a codon optimization process so that the gene can be expressed in E. coli.

(6) 2-2. Construction of a Plasmid Expressing metX Gene Derived from Sideroxydans lithotrophicus ES-1

(7) The metX gene was amplified by PCR using the primers of SEQ ID NOS: 4 and 5 based on the synthesized nucleotide sequence of SEQ ID NO: 3. The primer of SEQ ID NO: 5 has the HindIII restriction site.

(8) TABLE-US-00001 SEQIDNO:4) 5-ATCTTGAGTATTTCGGTTGGTATTG-3 SEQIDNO:5) 5-CCCAAGCTTttaagcagctgattcccaagc-3

(9) The PCR was performed for 30 cycles consisting of denaturation at 95 C. for 30 s, annealing at 55 C. for 30 s, and extension at 72 C. for 1 m. The PCR products were subjected to electrophoresis in a 1.0% agarose gel, and a 1.14 kb band was eluted, purified, and treated with HindIII. The pCL1920 vector including the cysK promoter was treated with EcoRV and HindIII, and the resulting restriction fragments were cloned. The plasmid expressing metX gene obtained as a result of the cloning was named as pCL-PcysK-metX (sli).

EXAMPLE 3

Construction of Experimental Strains

(10) 3-1. Deletion of metB Gene

(11) The metB gene encoding cystathionine gamma synthase in an wild type E. coli (K12) W3110 strain was deleted. For the deletion of the metB gene, the FRT-one-step-PCR deletion method was performed (PNAS (2000) vol 97: P 6640-6645). For the deletion of the metB gene, a deletion cassette was constructed by PCR using the primers of SEQ ID NOS: 6 and 7, and the pKD3 vector (PNAS (2000) vol 97: P 6640-6645) as a template.

(12) TABLE-US-00002 SEQIDNO:6) 5-TTACTCTGGTGCCTGACATTTCACCGACAAAGCCCAGGGAACTTCAT CACGTGTAGGCTGGAGCTGCTTC-3 SEQIDNO:7) 5-CGCTGCGCCAGCTCCATACGCGGCACCAGCGTTCGCAACCCACGTAG CAGCATATGAATATCCTCCTTAG-3

(13) The PCR was performed for 30 cycles consisting of denaturation at 95 C. for 30 s, annealing at 55 C. for 30 s, and extension at 72 C. for 1 m. The PCR products were subjected to electrophoresis in a 1.0% agarose gel, and a 1.1 kb band was eluted and purified. The recovered DNA fragment was electroporated into the E. coli (K12) W3110 strain, which was already transformed with the pKD46 vector (PNAS (2000) vol 97 P 6640-6645). For the electroporation, the W3110 strain, which was transformed with the pKD46, was cultured in LB medium containing 200 g/L of ampicillin and 5 mM L-arabinose at 30 C. until OD.sub.600 reached 0.5, and washed 3 times with 10% glycerol for use. The electroporation was performed at 2500V. The recovered strain was plated on LB plate medium containing 30 g/L chloramphenicol, cultured at 37 C. for 1 to 2 days, and the strain showing resistance to chloramphenicol was selected. The selected strain was subjected to PCR under the same conditions described above using the primers of SEQ ID NOS: 8 and 9, and the deletion of metB gene was confirmed by observing the presence of a 1.5 kb band of the gene in a 1.0% agarose gel.

(14) TABLE-US-00003 SEQIDNO:8) 5-TATTCGCCGCTCCATTCAGC-3 SEQIDNO:9) 5-TACCCCTTGTTTGCAGCCCG-3

(15) The thus-confirmed strain was transformed with the pCP20 vector (PNAS (2000) vol 97 P 6640-6645) and cultured in LB medium containing 100 g/L of ampicillin. The final strain with a deletion of metB gene having a reduced size, which was confirmed in a 1.0% agarose gel, was constructed by performing PCR under the same conditions and confirmed that the chloramphenicol marker was removed from the strain. The thus-constructed strain, which requires methionine, was named as CC03-0131.

(16) 3-2. Deletion of thrB Gene

(17) The amount of synthesis of O-succinylhomoserine from homoserine was attempted to increase by deleting the thrB gene which encodes homoserine kinase. In particular, it is essential to delete the thrB gene to use a threonine-producing strain, because the utilization activity of homoserine is very high. The deletion of the thrB gene in the CC03-0131 strain constructed above was performed by the FRT-one-step-PCR deletion method. A thrB deletion cassette was constructed by PCR using the primers of SEQ ID NOS: 10 and 11 and the pKD3 vector as a template.

(18) TABLE-US-00004 SEQIDNO:10) 5-CATGGTTAAAGTTTATGCCCCGGCTTCCAGTGCCAATATGAGCGTCG GGTGTGTAGGCTGGAGCTGCTTC-3 SEQIDNO:11) 5-GGAGATACCGCTCGCTACCGCGCCGATTTCCGCGACCGCCTGCCGCG CCTCATATGAATATCCTCCTTAG-3

(19) The PCR was performed for 30 cycles consisting of denaturation at 95 C. for 30 s, annealing at 55 C. for 30 s, and extension at 72 C. for 1 m. The PCR products were subjected to electrophoresis in a 1.0% agarose gel, and a 1.1 kb band was eluted and purified. The recovered DNA fragment was electroporated into the CC03-0131 strain, which was already transformed with the pKD46 vector. For the electroporation, the CC03-0131 strain, which was transformed with the pKD46, was cultured in LB medium containing 200 g/L of ampicillin and 5 mM arabinose at 30 C. until OD.sub.600 reached 0.5, and washed 3 times with 10% glycerol for use. The electroporation was performed at 2500V. The recovered strain was plated on LB plate medium containing 30 g/L chloramphenicol, cultured at 37 C. for 1 to 2 days, and the strain showing resistance to chloramphenicol was selected.

(20) The selected strain was subjected to PCR under the same conditions described above using the primers of SEQ ID NOS: 12 and 13, and the deletion of thrB gene was confirmed by observing the presence of a 1.5 kb band of the gene in a 1.0% agarose gel.

(21) TABLE-US-00005 SEQIDNO:12) 5-ACTCGACGATCTCTTTGCC-3 SEQIDNO:13) 5-ACGCCGAGAGGATCTTCGCAG-3

(22) The thus-confirmed strain was transformed with the pCP20 vector and cultured in LB medium containing 100 g/L of ampicillin. The final strain with a deletion of thrB gene having a reduced size, which was confirmed in a 1.0% agarose gel, was constructed by performing PCR under the same conditions and confirmed that the chloramphenicol marker was removed from the strain. The thus-constructed strain was named as CC03-0131-2.

(23) 3-3. Deletion of metA Gene

(24) For the characterization of substrate specificity and activity of the metX gene derived from Sideroxydans lithotrophicus ES-1 in an E. coli strain, the original metA gene on the chromosome was deleted based on the CC03-0131-2 strain, which is an E. coli (K12) W3110 strain with deletions of metB and thrB genes. The metA gene was deleted by the FRT-one-step-PCR deletion method. A metA deletion cassette was constructed by PCR using the primers of SEQ ID NOS: 14 and 15 and the pKD3 vector as a template.

(25) TABLE-US-00006 SEQIDNO:14) 5-TCAGCTGTTGCGCATCGATTCCCGTGAATCGCGCAACACGCCCGCAG AGCGTGTAGGCTGGAGCTGCTTC-3 SEQIDNO:15) 5-CCGTCACAAAGGCAATGCGCTTATCTTTACTGGCAAACAGATATGCA TCCCATATGAATATCCTCCTTAG-3

(26) The PCR was performed for 30 cycles consisting of denaturation at 95 C. for 30 s, annealing at 55 C. for 30 s, and extension at 72 C. for 1 m. The PCR products were subjected to electrophoresis in a 1.0% agarose gel, and a 1.1 kb band was eluted and purified. The recovered DNA fragment was electroporated into the CC03-0131-2 strain, which was already transformed with the pKD46 vector. For the electroporation, the CC03-0131-2 strain, which was transformed with the pKD46, was cultured in LB medium containing 200 g/L of ampicillin and 5 mM arabinose at 30 C. until OD.sub.600 reached 0.5, and washed 3 times with 10% glycerol for use. The electroporation was performed at 2500V. The recovered strain was plated on LB plate medium containing 30 g/L chloramphenicol, cultured at 37 C. for 1 to 2 days, and the strain showing resistance to chloramphenicol was selected.

(27) The selected strain was subjected to PCR under the same conditions described above using the primers of SEQ ID NOS: 16 and 17, and the deletion of metA gene was confirmed by observing the presence of a 1.5 kb band of the gene in a 1.0% agarose gel.

(28) TABLE-US-00007 SEQIDNO:16) 5-CTCATTAACGTTGGTTGTCA-3 SEQIDNO:17) 5-TATCTTGCTGCTGCTGAATG-3

(29) The thus-confirmed strain was transformed with the pCP20 vector and cultured in LB medium containing 100 g/L of ampicillin. The final strain with a deletion of metA gene having a reduced size, which was confirmed in a 1.0% agarose gel, was constructed by performing PCR under the same conditions and confirmed that the chloramphenicol marker was removed from the strain. The thus-constructed strain was named as CC03-0132.

(30) 3-4. Construction of a Strain Introduced with a Plasmid Expressing metX Gene Derived from Sideroxydans lithotrophicus ES-1

(31) For the characterization of substrate specificity and activity of the metX gene derived from Sideroxydans lithotrophicus ES-1, the CC03-0132 strain, which is an E. coli (K12) W3110 strain with deletions of metB, thrB and metA genes, was introduced with the plasmid pCL-PcysK-metX (sli) constructed in Example 2.

(32) The CC03-0132 strain introduced with the pCL-PcysK-metX (sli) was named as CC03-0136 and deposited at the Korean Culture Center of Microorganisms (KCCM) located at 361-221, Hongje-l-dong, Seodaemun-gu, Seoul, Korea, which is a subsidiary of the Korean Federation of Culture Collections (KFCC), recognized as an international depositary authority under the Budapest Treaty, on Jun. 10, 2013 under the Accession Number KCCM11424P.

(33) A strain was constructed by introducing a plasmid pCL-PcysK-metA, which was constructed in the same manner as in Example 2 except that using wild type metA in the CC03-0132 strain as the control group. The thus-constructed strain was named as CC03-0132/pCL-PcysK-metA.

(34) Additionally, a strain was constructed using a threonine-producing strain CJM002, which was released from the methionine requirement (Accession Number: KCCM-10568), via artificial mutation using NTG based on the L-threonine-producing strain TF4076 (Accession Number: KFCC-10718), which is a methionine-requiring strain disclosed in Korean Pat. No. 10-0905381, in the same manner as in Examples 3-1 to 3-3, and the thus-constructed strain was named as CJM-BTA.

(35) The plasmids, pCL-PcysK-metX(sli) and pCL-PcysK-metA, as described above, were introduced based on the CJM-BTA strain, and the thus-constructed strain was named as CJM-BTA/pCL-PcysK-metX (sli) and CJM-BTA/pCL-PcysK-metA, respectively.

EXAMPLE 4

Production of O-Succinylhomoserine Using a Strain

(36) 4-1. Flask Culture Experiment

(37) For the characterization of substrate specificity and activity of the metX gene derived from Sideroxydans lithotrophicus ES-1, introduced into the strain constructed in Example 3, an Erlenmeyer flask culture was performed. The composition of the flask culture is shown in Table 1 below.

(38) TABLE-US-00008 TABLE 1 Concentration Composition Stock (per Liter) Volume (mL) Glucose 40 g 200 KH.sub.2PO.sub.4 2 g 100 Ammonium sulfate 17 g 500 MgSO.sub.47H.sub.2O 1 g Yeast extract 4 g Methionine 0.4 g MnSO.sub.47H.sub.2O 10 mg/mL 0.01 g (1 mL of stock) ZnSO.sub.47H.sub.2O 1 mg/mL 0.01 g (10 mL of stock) FeSO.sub.47H.sub.2O 10 mg/mL 10 mg (1 mL of stock) Calcium carbonate 30 g 200

(39) The CC03-0132 strain and the CJM-BTA strain were inoculated into LB plate medium as control groups. The CC03-0136 strain (transformed with a metX expression vector), the CC03-0132/pCL-PcysK-metA strain (transformed with the metX expression vector prepared using the same vector), and two other strains, CJM-BTA/pCL-PcysK-metX (sli) and CJM-BTA/pCL-PcysK-metA (transformed with the metX expression vector or the metA expression vector based on the CJM-BTA strain, respectively) were inoculated into LB plate media containing spectinomycin, cultured at 33 C. overnight. Then, single colonies were inoculated into 2 mL of LB medium containing spectinomycin, cultured at 33 C. for 2 hours, inoculated again into a 250 mL Erlenmeyer containing 25 mL of flask medium to an absorbance of 0.07 at OD600, cultured at 33 C. at a rate of 200 rpm for 48 hours, and the amount of O-succinylhomoserine production was compared via HPLC analysis. The results are shown in Table 2 below.

(40) TABLE-US-00009 TABLE 2 Glucose Amount of consumption O-succinylhomoserine Strain OD (g/L) production (g/L) CC03-0132 16 40 0.0 CC03-0132/ 20 40 0.5 pCL-PcysK-metA CC03-0132/ 20 40 1.3 pCL-PcysK-metX (sli): CC03-0136 CJM-BTA 5 40 0.0 CJM-BTA/ 6 40 1.0 pCL-PcysK-metA CJM-BTA/ 6 40 3.5 pCL-PcysK-metX (sli)

(41) As a result, it was confirmed that the metX gene derived from Sideroxydans lithotrophicus ES-1, as is the case with the metA gene of E. coli, produces O-succinylhomoserine using succinyl-CoA as a substrate but did not produce O-acetylhomoserine. When the metX gene derived from Sideroxydans lithotrophicus ES-1 was introduced, no feedback inhibition by the methionine added to the medium appeared even with the wild type itself without introduction of any modification.

(42) Those of ordinary skill in the art will recognize that the present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the present invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within the scope of the present invention.