MICROORGANISM HAVING IMPROVED INTRACELLULAR ENERGY LEVEL AND METHOD FOR PRODUCING L-AMINO ACID USING SAME

Abstract

The present application relates to a recombinant microorganism having an improved intracellular energy level and a method for producing L-amino acid using the microorganism.

Claims

1. A microorganism of the genus Escherichia having an increased intracellular ATP level, compared to an unmodified strain, wherein activities of one or more proteins selected from an amino acid sequence of SEQ ID NO: 5, an amino acid sequence of SEQ ID NO: 6, and an amino acid sequence of SEQ ID NO: 7, which constitute an iron uptake system, were inactivated.

2. The microorganism of the genus Escherichia according to claim 1, wherein the activities of all of the proteins having amino acid sequences of SEQ ID NOS: 5, 6, and 7 were inactivated.

3. The microorganism of the genus Escherichia according to claim 1, wherein the microorganism is E. coli.

4. The microorganism of the genus Escherichia according to claim 1, wherein the microorganism of the genus Escherichia has an improved ability to produce L-amino acid, compared to an unmodified strain.

5. The microorganism of the genus Escherichia according to claim 4, wherein the L-amino acid is L-threonine or L-tryptophan.

6. A method for producing L-amino acids, the method comprising: culturing the microorganism of the genus Escherichia of claim 1 in a media, and recovering L-amino acids from the culture media or the microorganism.

7. The method of claim 6, wherein the L-amino acid is L-threonine or L-tryptophan.

8. A method for producing L-amino acids, the method comprising: culturing the microorganism of the genus Escherichia of claim 2 in a media, and recovering L-amino acids from the culture media or the microorganism.

9. A method for producing L-amino acids, the method comprising: culturing the microorganism of the genus Escherichia of claim 3 in a media, and recovering L-amino acids from the culture media or the microorganism.

10. A method for producing L-amino acids, the method comprising: culturing the microorganism of the genus Escherichia of claim 4 in a media, and recovering L-amino acids from the culture media or the microorganism.

11. A method for producing L-amino acids, the method comprising: culturing the microorganism of the genus Escherichia of claim 5 in a media, and recovering L-amino acids from the culture media or the microorganism.

12. The method of claim 8, wherein the L-amino acid is L-threonine or L-tryptophan.

13. The method of claim 9, wherein the L-amino acid is L-threonine or L-tryptophan.

14. The method of claim 10, wherein the L-amino acid is L-threonine or L-tryptophan.

15. The method of claim 11, wherein the L-amino acid is L-threonine or L-tryptophan.

Description

DESCRIPTION OF THE DRAWINGS

[0036] FIG. 1 shows intracellular ATP levels of E. coli according to a specific embodiment of the present application, compared to an unmodified strain;

[0037] FIG. 2 shows intracellular ATP levels of wild-type-derived E. coli having a producibility of L-tryptophan according to a specific embodiment of the present application, compared to an unmodified strain;

[0038] FIG. 3 shows intracellular ATP levels of E. coli having a producibility of L-threonine according to a specific embodiment of the present application, compared to an unmodified strain;

[0039] FIG. 4 shows intracellular ATP levels of E. coli having a producibility of L-tryptophan according to a specific embodiment of the present application, compared to an unmodified strain;

[0040] FIG. 5 shows L-threonine producibility of E. coli having a producibility of L-threonine according to a specific embodiment of the present application, compared to an unmodified strain; and

[0041] FIG. 6 shows L-tryptophan producibility of E. coli having a producibility of L-tryptophan according to a specific embodiment of the present application, compared to an unmodified strain.

MODE OF THE INVENTION

[0042] Hereinafter, the present application will be described in more detail with reference to Examples. However, these Examples are for illustrative purposes only, and the scope of the present application is not intended to be limited by these Examples.

Example 1

Preparation of Wild-Type E. coli W3110 Having Inactivation of Proteins Encoded by fhuC, fhuD, and fhuB Genes

[0043] In this Example, fhuC, fhuD, and fhuB genes of wild-type E. coli W3110 (ATCC 39936) were deleted by homologous recombination, respectively.

[0044] The fhuC, fhuD, and fhuB genes to be deleted have nucleotide sequences of SEQ ID NOs: 1, 2, and 3, respectively and these genes exist in the form of operon of SEQ ID NO: 4.

[0045] To delete fhuC, fhuD, and fhuB, one-step inactivation using lambda Red recombinase developed by Datsenko K A, et al. was performed (Proc Natl Acad Sci USA., (2000) 97:6640-6645). As a marker to confirm the insertion into the gene, a chloramphenicol gene of pUCprmfmloxC, which was prepared by ligating an rmf promoter to pUC19 (New England Biolabs (USA)) and ligating a mutated loxP-CmR-loxP cassette obtained from pACYC184 (New England Biolab) thereto, was used (Korean Patent Application NO. 2009-0075549).

[0046] First, primary polymerase chain reaction (hereinbelow, referred to as PCR) was performed using pUCprmfmloxC as a template and primer combinations of SEQ ID NOs: 8 and 9, 10 and 11, 12 and 13, and 8 and 13 having a part of the fhuC and fhuB genes and a partial sequence of the chloramphenicol resistant gene of the pUCprmfmloxC gene under the conditions of 30 cycles of denaturation at 94 C. for 30 seconds, annealing at 55 C. for 30 seconds and elongation at 72 C. for 1 minute, thereby obtaining PCR products of about 1.2 kb, fhuC1st, fhuD1st, fhuB1st, and fhuCDB1st.

[0047] Thereafter, the PCR products of 1.2 kb, fhuC1st, fhuD1st, fhuB1st, and fhuCDB1st obtained by PCR were electrophoresed on a 0.8% agarose gel, and then eluted and used as a template for secondary PCR. The secondary PCR was performed using the eluted primary PCR products as templates and the primer combinations of SEQ ID NOs: 14 and 15, 16 and 17, 18 and 19, 14 and 19 containing nucleotide sequences of 20 bp of the 5 and 3 regions of the PCR products obtained in the primary PCR under the conditions of 30 cycles of denaturation at 94 C. for 30 seconds, annealing at 55 C. for 30 seconds and elongation at 72 C. for 1 minute, thereby obtaining PCR products of about 1.3 kb, fhuC, fhuD, fhuB, and fhuCDB. The PCR products thus obtained was electrophoresed on a 0.8% agarose gel, and then eluted, and used in recombination.

[0048] E. coli W3110, which was transformed with a pKD46 vector according to the one-step inactivation method developed by Datsenko K A et al. (Proc Natl Acad Sci USA., (2000) 97:6640-6645), was prepared as a competent strain, and transformation was performed by introducing the gene fragment of 1.3 kb obtained by primary and secondary PCR. The strains were cultured on the LB medium supplemented with chloramphenicol and transformants having chloramphenicol resistance were selected. Deletion of any or all of fhuC, fhuD, and fhuB was confirmed by PCR products of about 4.4 kb, about 4.3 kb, about 3.3 kb, and about 1.6 kb which were amplified by PCR using genomes obtained from the selected strains as templates and primers of SEQ ID NOs: 20 and 21.

[0049] After removal of pKD46 from the primary recombinant strains having chloramphenicol resistance thus obtained, a pJW168 vector (Gene, (2000) 247, 255-264) was introduced into the primary recombinant strains having chloramphenicol resistance so as to remove the chloramphenicol marker gene from the strains (Gene, (2000) 247, 255-264). PCR was performed using primers of SEQ ID NOs: 20 and 21 to obtain PCR products of about 3.4 kb, about 3.3 kb, about 2.2 kb, and about 0.6 kb, indicating that the strains finally obtained had deletion of any or all of fhuC, fhuD, and fhuB genes. The strains were designated as E. coli W3110_fhuC, W3110_fhuD, W3110_fhuB and W3110_fhuCDB, respectively.

Example 2

Measurement of Intracellular ATP Levels in Wild-Type E. coli-Derived fhuC, fhuD, and fhuB Gene-Deleted E. coli

[0050] In this Example, the intracellular ATP levels in the strains prepared in Example 1 were practically measured.

[0051] For this purpose, An Efficient Method for Quantitative determination of Cellular ATP Synthetic Activity developed by Kiyotaka Y. Hara et al., in which luciferase is used, was employed (J Biom Scre, (2006) Vol. 11, No. 3, pp 310-17). Briefly, E. coli W3110 which is an unmodified strain used in Example 1 and E. coli W3110_fhuCDB obtained by gene deletion were cultured overnight in LB liquid medium containing glucose, respectively. After culturing, supernatants were removed by centrifugation, the cells thus obtained were washed with 100 mM Tris-Cl (pH 7.5), and then treated with PB buffer (permeable buffer: 40% [v/v] glucose, 0.8% [v/v] Triton X-100) for 30 minutes to release intracellular ATP from the cells. Next, supernatants were removed by centrifugation, and luciferin as a substrate for luciferase was added to the cells. The cells were allowed to react for 10 minutes. Color development by luciferase was measured using a luminometer to quantitatively determine ATP levels. The results are given in FIG. 1. The results of FIG. 1 were recorded as the average of three repeated experiments.

[0052] As shown in FIG. 1, the intracellular ATP levels in E. coli W3110_fhuC, W3110_fhuD, W3110_fhuB and W3110_fhuCDB prepared in Example 1, in which any or all of wild-type E. coli-derived fhuC, fhuD, and fhuB was/were deleted, were increased, compared to the unmodified strain, E. coli W3110.

Example 3

Preparation of Wild-Type-Derived L-Tryptophan-Producing Strain Having Inactivation of Proteins Encoded by fhuC, fhuD, and fhuB Genes and Measurement of Intracellular ATP Levels

[0053] In this Example, any or all of fhuC, fhuD, and fhuB genes of a wild-type-derived L-tryptophan-producing strain, E. coli W3110 trp2/pCL-Dtrp_att-trpEDCBA (Korean Patent Publication No. 10-2013-0082121) was/were deleted by homologous recombination as in Example 1 to prepare W3110 trp2_fhuC/pCL-Dtrp_att-trpEDCBA, W3110 trp2_fhuD/pCL-Dtrp_att-trpEDCBA, W3110 trp2_fhuB/pCL-Dtrp_att-trpEDCBA, and W3110 trp2_fhuCDB/pCL-Dtrp_att-trpEDCBA strains. In these strains thus prepared, intracellular ATP levels were measured in the same manner as in Example 2 and the results are given in FIG. 2.

[0054] As shown in FIG. 2, the intracellular ATP levels in the strains, which were prepared by deletion of any or all of fhuC, fhuD, and fhuB genes in the wild-type L-tryptophan-producing strain, were increased, compared to an unmodified strain and a control strain.

Example 4

Examination of Titer of Wild-Type-Derived L-Tryptophan-Producing Strain Having Inactivation of Proteins Encoded by fhuC, fhuD, and fhuB Genes

[0055] As described in Example 3, the wild-type-derived L-tryptophan-producing strain, W3110 trp2/pCL-Dtrp_att-trpEDCBA and the strains with improved intracellular ATP levels prepared by deletion of any or all of fhuC, fhuD, and fhuB genes were subjected to titration using glucose as a carbon source.

[0056] Each of the strains was inoculated by a platinum loop on an LB solid medium, and cultured in an incubator at 37 C. overnight, and then inoculated by a platinum loop into 25 mL of a glucose-containing titration medium containing a composition of Table 1. Then, the strains were cultured in an incubator at 37 C. and at 200 rpm for 48 hours. The results are given in Table 2. All the results were recorded as the average of three repeated experiments.

TABLE-US-00001 TABLE 1 Concentration Composition (per liter) Glucose 60 g K.sub.2HPO.sub.4 1 g (NH.sub.4).sub.2SO.sub.4 15 g NaCl 1 g MgSO.sub.4H.sub.2O 1 g Sodium citrate 5 g Yeast extract 2 g CaCO.sub.3 40 g L-Phenylalanine 0.15 g L-tyrosine 0.1 g pH 6.8

TABLE-US-00002 TABLE 2 Production amount of L-tryptophan Strain (mg/L)* W3110 trp2/pCL-Dtrp_att-trpEDCBA 562 W3110 trp2_fhuC/pCL-Dtrp_att-trpEDCBA 781 W3110 trp2_fhuD/pCL-Dtrp_att-trpEDCBA 816 W3110 trp2_fhuB/pCL-Dtrp_att-trpEDCBA 779 W3110 trp2_huCDB/pCL-Dtrp_att-trpEDCBA 796 *measured at 48 hours

[0057] As shown in Table 2, it was demonstrated that the strains with improved intracellular ATP levels, prepared in Example 3 by deleting any or all of fhuC, fhuD, and fhuB genes in the wild-type L-tryptophan-producing strain W3110 trp2/pCL-Dtrp_att-trpEDCBA, increased L-tryptophan production up to about 63%, compared to the unmodified strain trp2/pCL-Dtrp_att-trpEDCBA. In view of the intracellular ATP levels confirmed in FIG. 2, these results indicate that L-tryptophan producibilities of the strains were increased by the increased intracellular ATP levels.

Example 5

Preparation of L-Threonine-Producing Strain and L-Tryptophan Producing Strain Having Inactivation of Proteins Encoded by fhuC, fhuD, and fhuB Genes

[0058] In this Example, fhuC, fhuD, and fhuB genes of the L-tryptophan producing strain KCCM10812P (Korean Patent No. 0792095) and the L-threonine producing strain KCCM10541 (Korean Patent No. 0576342) were deleted by homologous recombination, respectively, as in Example 1.

[0059] The unmodified strain having L-tryptophan producibility, E. coli KCCM10812P is a strain derived from an E. coli variant (KFCC 10066) having L-phenylalanine producibility, and is a recombinant E. coli strain having L-tryptophan producibility, characterized in that chromosomal tryptophan auxotrophy was desensitized or removed, pheA, trpR, mtr and tnaAB genes were attenuated, and aroG and trpE genes were modified.

[0060] Also, the unmodified strain having L-threonine producibility, E. coli KCCM10541P is a strain derived from E. coli KFCC10718 (Korean Patent Publication No. 1992-0008365), and is E. coli having resistance to L-methionine analogue, a methionine auxotroph phenotype, resistance to L-threonine analogue, a leaky isoleucine auxotroph phenotype, resistance to L-lysine analogue, and resistance to -aminobutyric acid, and L-threonine producibility.

[0061] The fhuC, fhuD, and fhuB genes to be deleted were deleted from E. coli KCCM10812P and E. coli KCCM10541P in the same manner as in Example 1, respectively. As a result, an L-threonine producing strain, KCCM10541_fhuCDB and an L-tryptophan producing strain, KCCM10812P_fhuCDB were prepared.

Example 6

Measurement of ATP Levels in L-Threonine Producing Strain and L-Tryptophan Producing Strain Having Inactivation of Proteins Encoded by fhuC, fhuD, and fhuB Genes

[0062] In this Example, the intracellular ATP levels in the strains prepared in Example 5 were practically measured.

[0063] The intracellular ATP levels were measured in the same manner as in Example 2. The results are given in FIGS. 3 and 4. The results of FIGS. 3 and 4 were recorded as the average of three repeated experiments. As control groups, used were ysa and ydaS-deleted, L-threonine producing strain (E. coli KCCM10541P_ysaydaS) and L-tryptophan producing strain (E. coli KCCM10812P_ysaydaS) which are known to have higher intracellular ATP levels than the unmodified strains, E. coli KCCM10812P and E. coli KCCM10541P used in Example 3 (Korean Patent No. 1327093).

[0064] As shown in FIGS. 3 and 4, fhuC, fhuD, and fhuB-deleted strains prepared from the L-threonine producing strain and the L-tryptophan producing strain in Example 3 showed increased intracellular ATP levels, compared to the unmodified strains and control strains.

Example 7

Examination of Titer of L-Threonine-Producing Strain Having the Inactivated Proteins Encoded by fhuC, fhuD, and fhuB Genes

[0065] As described in Example 5, the strains with improved intracellular ATP levels, which were prepared by deletion of fhuC, fhuD, and fhuB genes in an L-threonine producing microorganism, E. coli KCCM10541P (Korean Patent No. 0576342), were subjected to titration using glucose as a carbon source. The ysa and ydaS-deleted L-threonine producing strain (E. coli KCCM10541P_ysaydaS) was used as a control group to compare the titration results.

[0066] Each of the strains was cultured on an LB solid medium in an incubator at 33 C. overnight, and then inoculated by a platinum loop into 25 mL of a glucose-containing titration medium containing the composition of Table 3. Then, the strains were cultured in an incubator at 33 C. and at 200 rpm for 50 hours. The results are given in Table 4 and FIG. 5. All the results were recorded as the average of three repeated experiments.

TABLE-US-00003 TABLE 3 Concentration Composition (per liter) Glucose 70 g KH.sub.2PO.sub.4 2 g (NH.sub.4).sub.2SO.sub.4 25 g MgSO.sub.4H.sub.2O 1 g FeSO.sub.4H.sub.2O 5 mg MnSO.sub.4H.sub.2O 5 mg Yeast extract 2 g CaCO.sub.3 30 g

TABLE-US-00004 TABLE 4 Production Glucose amount of consumption L-threonine Strain OD.sub.562 (g/L)* (g/L)** KCCM10541P 22.8 41.0 28.0 0.5 KCCM10541P_ysaAydaS 23.9 42.1 29.8 0.9 KCCM10541P_fhuCDB 23.1 41.8 30.5 1.sup. *measured at 30 hours **measured at 50 hours

[0067] As shown in Table 4, it was demonstrated that the recombinant L-threonine producing E. coli strain prepared according to the present application showed a physiological activity similar to that of the unmodified strain, and increased L-threonine production up to about 9%, compared to the unmodified strain. In view of the intracellular ATP levels confirmed in FIG. 3, these results indicate that L-threonine producibilities of the strains were increased by the increased intracellular ATP levels.

Example 8

Examination of Titer of L-Tryptophan-Producing Strain Having Inactivation of Proteins Encoded by fhuC, fhuD, and fhuB Genes

[0068] As described in Example 5, the strains with improved intracellular ATP levels, which were prepared by deletion of fhuC, fhuD, and fhuB genes in an L-tryptophan producing microorganism, KCCM10812P (Korean Patent No. 0792095), were subjected to titration using glucose as a carbon source. The ysa and ydaS-deleted L-tryptophan producing strain (E. coli KCCM10812P_ysaydaS) was used as a control group to evaluate the titer in the same manner as in Example 4.

[0069] The titration results are given in Table 5 and FIG. 6. All the results were recorded as the average of three repeated experiments.

TABLE-US-00005 TABLE 5 Production Glucose amount of consumption L-tryptophan Strain OD.sub.600 (g/L)* (g/L)** KCCM10812P 18.2 45.7 5.5 0.2 KCCM10812P_ysaAydaS 18.3 46.3 6.7 0.1 KCCM10812P_fhuCDB 17.9 47.4 7.1 0.5 *measured at 33 hours **measured at 48 hours

[0070] As shown in Table 5, it was demonstrated that the recombinant L-tryptophan producing E. coli strain prepared according to the present application showed a physiological activity similar to that of the unmodified strain, and increased L-tryptophan production up to about 30%, compared to the unmodified strain. In view of the intracellular ATP levels confirmed in FIG. 4, these results indicate that L-tryptophan producibilities of the strains were increased by the increased intracellular ATP levels.

[0071] The recombinant strain of the present application, CA04-2801 (KCCM10812P_fhuCDB) was deposited at the Korean Culture Center of Microorganisms, an international depository authority, on Nov. 15, 2013 under Accession NO. KCCM11474P.

[0072] Based on the above description, it will be understood by those skilled in the art that the present application may be implemented in a different specific form without changing the technical spirit or essential characteristics thereof. Therefore, it should be understood that the above embodiment is not limitative, but illustrative in all aspects. The scope of the application is defined by the appended claims rather than by the description preceding them, and therefore all changes and modifications that fall within metes and bounds of the claims, or equivalents of such metes and bounds are therefore intended to be embraced by the claims.