MICROORGANISM OF THE GENUS ESCHERICHIA PRODUCING L-TRYPTOPHAN AND METHOD FOR PRODUCING L-TRYPTOPHAN USING THE SAME

Abstract

The present application relates to a microorganism of the genus Escherichia producing L-tryptophan and, more specifically, to a microorganism of the genus Escherichia with improved activity of producing L-tryptophan by weakening or inactivating the activity of endogenous 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase.

Additionally, the present application relates to a method for producing L-tryptophan using the microorganism of the genus Escherichia.

Claims

1. A microorganism of the genus Escherichia producing L-tryptophan, wherein the activities of endogenous 6-phosphogluconate dehydratase (Edd) and 2-keto-3-deoxy-6-phosphogluconate aldolase (Eda) are weakened or inactivated.

2. The microorganism according to claim 1, wherein the 6-phosphogluconate dehydratase has an amino acid sequence set forth in SEQ ID NO: 1.

3. The microorganism according to claim 1, wherein the 2-keto-3-deoxy-6-phosphogluconate aldolase has an amino acid sequence set forth in SEQ ID NO: 3.

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

5. The microorganism according to claim 1, wherein an entirety or a part of the pheA gene, trpR gene, mtr gene, and tnaAB gene is further deleted.

6. A method of preparing L-tryptophan, comprising: culturing the microorganism of the genus Escherichia of any of claims 1 to 5 in a medium; and recovering L-tryptophan from the cultured medium or the cultured microorganism.

Description

EXAMPLE 1

Preparation of a eddeda Strain from the Parent Strain of Comparative Example 1

[0054] In the wild-type strain W3110 pheAtrpRmtrtnaAB/pCL1920-Ptrc-trpO prepared in Comparative Example 1, the edd-eda gene group was deleted simultaneously by homologous recombination, and thereby a strain, in which both 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate, encoded by the edd gene (SEQ ID NO: 2) and the eda gene (SEQ ID NO: 4), were both inactivated, was prepared.

[0055] Specifically, for the preparation of the above strain, the one-step inactivation method, which is a mutant-generating technology using lambda Red recombinase developed by Datsenko K A et al., was used. As the marker for confirming the insertion into a gene, chloramphenicol gene of pUCprmfmloxC was used (Korean Patent Application Publication No. 2009-007554). About 1,200 pairs of gene fragments were amplified by PCR by repeating 30 cycles of denaturation at 94 C. for 30 seconds, annealing at 55 C. for 30 seconds, and polymerization at 72 C. for 1 minute, using pUCprmfmloxC as a template along with primers 27 and 28 having a part of the edd-eda gene group and a partial nucleotide sequence of the chloramphenicol-resistant gene of the pUCprmfmloxC gene.

[0056] Additionally, the DNA fragments obtained by PCR amplification were electrophoresed in a 0.8% agarose gel, eluted, and used as a template for the second PCR. The second PCR was designed to obtain 20 pairs of complementary nucleotide sequences in the 5 and 3 regions of the first DNA fragments, and about 1,300 pairs of gene fragments were amplified by PCR by repeating 30 cycles of denaturation at 94 C. for 30 seconds, annealing at 55 C. for 30 seconds, and polymerization at 72 C. for 1 minute, using the first PCR product as a template along with primers 29 and 30, to which the 5 and 3 regions of the edd-eda gene group were added. The thus-obtained DNA fragments were electrophoresed in a 0.8% agarose gel, eluted, and used for recombination.

[0057] The E. coli transformed with pKD46 according to the method developed by Datsenko K A et al. was prepared in a competent state, and the gene fragment with a size of 1,300 base pairs obtained by PCR was introduced therein for transformation. The thus-obtained strain was selected in LB medium containing chloramphenicol, and the PCR product obtained using primers 31 and 32 had a size of 1,626 base pairs, thus confirming that the edd-eda gene group was deleted.

[0058] The first recombinant strain having chloramphenicol resistance, after removing pKD46, was introduced with pJW 168, thereby removing chloramphenicol marker gene from the bacterial body (Gene, (2000) 247, 255-64). The finally-obtained bacterial body was a PCR-amplified product, obtained using primers 31 and 32, having a size of 580 pairs, thus confirming that the intended deletion was made. Additionally, after preparing the strain in a competent state, the strain was transformed by introducing the vector prepared in Comparative Example 1 therein, thereby preparing a W3110pheAtrpRmtrtnaABeddeda/pCL1920-Ptrc-trpO strain producing tryptophan.

EXAMPLE 2

Confirmation of the Tryptophan-Producing Ability of eddeda Strain

[0059] Titer evaluation was performed using the strain prepared in Comparative Example 1 and Example 1. For the titer evaluation, the bacterial body was inoculated with a platinum loop, cultured in solid LB medium overnight, and inoculated with a platinum loop onto each 25 mL flask titer medium having the composition shown in Table 1 below. After the inoculation, the strain was cultured at 37 C. at a rate of 200 rpm for 42 hours, and the results obtained therefrom are shown in Table 2 below. All results used represent the average value of the results obtained from three different flasks.

TABLE-US-00001 TABLE 1 Concentration Composition (per liter) Glucose 30 g K.sub.2HPO.sub.4 1 g (NH.sub.4).sub.2SO.sub.4 10 g NaCl 1 g MgSO.sub.47H.sub.2O 1 g Sodium citrate 5 g Yeast extract 2 g Calcium carbonate 40 g Sodium citrate 5 g Phenylalanine 0.15 g Tyrosine 0.1 g pH 6.8 g

TABLE-US-00002 TABLE 2 Glucose Consumption L-tryptophan Strain OD (g/L)* (g/L)** W3110pheAtrpRmtr 30.1 24.7 0.78 tnaAB/pCL1920-Ptrc-trpO Example 1 29.5 25.1 1.03 (W3110pheAtrpRmtrtnaAB eddeda/pCL1920-Ptrc-trpO) *value measured at the time-point of 22 hours **value measured at the time-point of 42 hours

[0060] As a result of the above experiment, as shown in Table 2 above, when the edd-eda gene group suggested in the present application was deleted, there was no significant difference in the glucose consumption compared to that of the parent strain in Comparative Example 1, however, the amount of tryptophan production was shown to increase by about 32% compared to that of the parent strain. This result is thought to be due to the fact that the reaction proceeded to ribulose 5-phosphate without the loss of 6-phosphogluconate, which is a substrate, by the deletion in the Entner Doudoroff pathway, and thus not only the amount of NADPH but also the amount of 5-phosphoribosyl-1-pyrophosphate (PRPP) and erythrose 4-phosphate (E4P) was increased, thereby improving the ability to produce tryptophan.

EXAMPLE 3

Preparation of a eddeda Strain from the Deposited Parent Strain

[0061] The L-tryptophan-producing E. coli KCCM11166P (Korean Patent No. 10-1261147) deposited in the Korean Culture Center of Microorganisms (KCCM) was treated in the same manner as in Example 1 and thereby a KCCM11166Peddeda strain, in which the edd-eda gene group was deleted, was prepared.

TABLE-US-00003 TABLE3 SEQ Primer ID Gene No. Sequence(5.fwdarw.3) NO pheA 1 AGGCAACACTATGACATCGTGTAGGCTGGAGCTGCTTC 16 2 GGTCGCCATTAACAACGTGGCATATGAATATCCTCCTTAG 17 3 TATTGAGTGTATCGCCAAC 18 4 CGATGTCATAGTGTTGCC 19 5 CCACGTTGTTAATGGCGACC 20 6 TTCATTGAACGGGTGATTTC 21 trpR 7 TCCGCACGTTTATGATATGCTATCGTACTCTTTAGCGAG 22 TACAACCGGGGGTGTAGGCTGGAGCTGCTTC 8 GCCACGTCTTATCAGGCCTACAAAATCAATCGCTTTTCA 23 GCAACACCTCTCATATGAATATCCTCCTTAG 9 GCGCCGGGCGTATCGACGCA 24 10 GCATATCATAAACGTGCGGA 25 11 TGTAGGCCTGATAAGACGTG 26 12 AAGGGGCGATCGGCGTGTTT 27 mtr 13 ATGGCAACACTAACCACCACCCAAACGTCACCGTCGCT 28 GCTTGGCGGCGTGTGTAGGCTGGAGCTGCTTC 14 TTACTGATACACCGGCAGTAAATTAAAGCTCGATAAAA 29 TATGCACCAGTGCATATGAATATCCTCCTTAG 15 GCAGCCGTTACATTGGTAAC 30 16 GTGGTGGTTAGTGTTGCCAT 31 17 TACTGCCGGTGTATCAGTAA 32 18 TCAAACCGTCAGCACGGCTG 33 tnaAB 19 ATGAAGGATTATGTAATGGAAAACTTTAAACATCTCCCT 34 GAACCGTTCCGGTGTAGGCTGGAGCTGCTTC 20 TTAGCCAAATTTAGGTAACACGTTAAAGACGTTGCCGA 35 ACCAGCACAAAACATATGAATATCCTCCTTAG 21 TTAAGCGAAATCACCGGGGAA 36 22 ATGTCCGAGCACTGGCGC 37 pCL1920- 23 CCCAAGCTTGCTGTTGACAATTAATCAT 38 Ptrc-trpO 24 AAAACTGCAGCTGTTTCCTGTGTGAAAT 39 25 AAAACTGCAGATGCAAACACAAAAACCGACT 40 26 AAAACTGCAGTTAACTGCGCGTCGCCGCTTTC 41 edd-eda 27 AAACGCGTTGTGAATCATCCTGCTCTGACAACTCAATTT 42 CAGGAGCCTTTGCCGCCAGCTGAAGCTTTAC 28 ACAGCACGCTTTTCAGCGCCAGGTAGTCACGGTAGTTA 43 GCCGGAGAAATATAGTGGATCTGATGGGTACC 29 TGCCCTATGAGCTCCGGTTACAGGCGTTTCAGTCATAAA 44 TCCTCTGAATGAAACGCGTTGTGAATCATCC 30 ATCGCCCGCTTCCAGCGCATCTGCCGGAACCAGCCAGG 45 AACCACCGATGCACAGCACGCTTTTCAGCGCC 31 CATGATCTTGCGCAGATTGTA 46 32 CATGATCTTGCGCAGATTGTA 47

EXAMPLE 4

Confirmation of the Tryptophan-Producing Ability of eddeda Strain

[0062] Titer evaluation was performed using the deposited strain KCCM11166P and the strain prepared in Example 3.

[0063] For the titer evaluation, the bacterial body was inoculated with a platinum loop, cultured in solid LB medium overnight, and inoculated with a platinum loop onto each 25 mL flask titer medium having the composition shown in Table 4 below. After the inoculation, the strain was cultured at 37 C. at a rate of 200 rpm for 42 hours, and the results obtained therefrom are shown in Table 5 below. All results used represent the average value of the results obtained from three different flasks.

TABLE-US-00004 TABLE 4 Concentration Composition (per liter) Glucose 60 g K.sub.2HPO.sub.4 1 g (NH.sub.4).sub.2SO.sub.4 10 g NaCl 1 g MgSO.sub.47H.sub.2O 1 g Sodium citrate 5 g Yeast extract 2 g Calcium carbonate 40 g Sodium citrate 5 g Phenylalanine 0.15 g Tyrosine 0.1 g pH 6.8 g

TABLE-US-00005 TABLE 5 Glucose Consumption L-tryptophan Strain OD (g/L)* (g/L)** KCCM11166P 22.9 31.0 5.7 KCCM11166Peddeda 23.6 30.7 6.9 *value measured at the time-point of 33 hours **value measured at the time-point of 48 hours

[0064] As a result of the above experiment, as shown in Table 5 above, when the edd-eda gene group suggested in the present application was deleted, there was no significant difference in the glucose consumption compared to that of the parent strain, however, the amount of tryptophan production was shown to increase by about 21% compared to that of the parent strain. This result is thought to be due to the improvement in the tryptophan-producing ability, as mentioned in Example 2 above.

[0065] The present inventors have confirmed that the KCCM11166P-based strain, in which the edd-eda gene group was inactivated, has an improved tryptophan-producing ability, named the strain as CA04-2800, and deposited it to the KCCM on Nov. 15, 2013, and it was assigned the deposit number KCCM11473P.

[0066] The above results suggest that the simultaneous inactivation of edd-eda activities in a microorganism of the genus Escherichia having the Entner-Doudoroff pathway can improve the tryptophan-producing ability, compared to the microorganism without the inactivation of edd-eda activities.

[0067] In the present application, the detailed description of those which can be sufficiently acknowledged and drawn by one of ordinary skill in the art is omitted, and various modifications, in addition to the exemplary embodiments described herein, may be included within the spirit and scope of the present application without modifying the technical concepts or essential characteristics of the present application. Accordingly, the present application may be embodied in other specific forms and one of ordinary skill in the art to which the present application pertains will be able to understand the same.

[Deposit Number]

[0068] Deposit Authority: Korean Culture Center of Microorganisms (overseas) [0069] Deposit Number: KCCM11473P [0070] Date of Deposit: 20131115