KR101653245B1 - Fermentation process for preparing thymidine by the recombinant E. coli - Google Patents

Abstract

A method for producing industrial grade thymidine in E. coli using a metabolic engineering approach is disclosed. The present invention relates to a gene having a nucleotide sequence of SEQ ID NO: 1, deoA , tdk , upp and udp , which are genes involved in a reuse metabolic pathway, is removed, a gene udhA having a nucleotide sequence of SEQ ID NO: 2, yfjB and the SEQ ID NO: gene pspB having the nucleotide sequence described by the three is expressed, carbamoyl-aspartate is Tate gene, purR, pepA and argR relating to the synthesis inhibition is removed and genes that make up the pyr operon (operon) pyrBI, pyrC , pyrD, and pyrE overexpressing the thymidine-producing E. coli strain.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for mass production of thymidine using a metabolic engineering method in Escherichia coli (Fermentation process for preparing thymidine by the recombinant E. coli)

The present invention relates to thymidine production using E. coli, and more particularly, to a method for mass-producing thymidine from a strain mutated through genetic recombination in E. coli in high purity.

The synthesis of nucleic acids using biological methods has been studied mainly as a precursor of food additives and various pharmaceutical preparations. In particular, pyrimidine deoxynucleoside such as thymidine or deoxyuridine synthesizes various antiviral therapeutic agents such as azidothymidine (AZT) and zidovudine It is known as a highly industrially useful precursor substance. Pyrimidine deoxynucleosides, including thymidine, are synthesized in all proliferating organisms, but their amount is strongly controlled and their concentration is low and limited, and so far they have been produced through low-cost, high-cost organic synthesis.

Many studies have been reported on a method for controlling thymidine metabolism and producing thymidine using microorganisms (see Non-Patent Document 1). They removed deoA and udp to prevent the degradation of thymidine and to remove tdk to inhibit the re-conversion of thymidine to dTMP (deoxythymidine monophosphate). Especially in E. coli , deoA And removal of udp alone results in almost no decomposition of thymidine. However, the coincident increase of other nucleosides such as uridine is problematic, which also affects the osmotic pressure, resulting in inhibition of cell growth (85-90%).

The thymidine biosynthesis process involves the reduction of oxygen from the ribose by NDP to deoxyribonucleoside diphosphate (NDP) by a ribonucleoside diphosphate reductase. Here, NADPH is used as a coenzyme to supply electrons. In addition to NADPH, thioredoxin and glutaredoxin act as electron donors. Therefore, the reinforcement of the reducing power can increase the biosynthesis of the main nucleic acid, for example, the expression of T4 phage is more effective than the introduction of thioredoxin, which is a foreign enzyme species having a high enzyme activity, to increase the expression level (see Non-Patent Documents 2 and 3 ). For this reason, studies have been carried out to reinforce this process in companies using cyimidine producing strains requiring high reducing power.

During the production of pyrimidine deoxynucleosides in E. coli, transcription and expression of the related enzymes are strongly regulated by various mechanisms. There are mechanisms such as feedback inhibition and attenuation depending on the concentration of (deoxy) nucleoside / (deoxy) nucleotide present (see Non-Patent Document 4). In order to overcome this, the gene involved in thymidine degradation was removed and dTMP phosphohydrolase was expressed, but studies on the necessary regulatory elements are still required to produce industrial-grade thymidine.

[Non-Patent Document]

Non-Patent Document 1: R. M. Q. Shanks, N. C. Caiazza, S. M. Hinsa, C. M. Toutain, G. A. O'Toole, 2006, Saccharomyces cerevisiae-Based Molecular Tool Kit for Manipulation of Genes from Gram-Negative Bacteria, Appl. Environ. Microbiol, 72 (7), 5027-5036.

Non-Patent Document 2: HC Lee, JH Kim, JS Kim, W. Jang, SY Kim, 2009, Fermentative production of thymidine by a metabolically engineering E. coli strain., Appl. Environ. Microbiol., 75 (8): 2423-2432.

NADPH / NADP + ratio improves thymidine production by ametabolically engineered strain, J. Biotechnol., 20; 149 (1-2): 24- 32.

Non-Patent Document 4: Neuhard, J. and RA Kelln, Biosynthesis and Conversion of Pyrimidines, Chapter 35 [In] Neidhardt, FC et al . [eds] " Escherichia coli and Salmonella Cellular and Molecular Biology ", Second Edition, Vol. I, pp 580-599, ASM Press, Washington DC, 1996.

The present invention thus provides a method for producing industrial grade thymidine in E. coli using a metabolic engineering approach.

In order to solve the above-mentioned problem, the present invention provides a recombinant vector comprising a gene udhA having the nucleotide sequence of SEQ ID NO: 1, deoA , tdk , upp and udp , which are genes involved in a reuse metabolic pathway, having the nucleotide sequence described by the gene yfjB and SEQ ID NO: 3 having the nucleotide sequence described gene is pspB expression, carbamoyl-aspartate is Tate gene, purR, pepA and argR relating to the synthesis inhibition is removed, pyr operon (operon ( Pyridine , pyrC , pyrD, and pyrE ), which constitute the thymidine-producing E. coli mutant strain.

Also, the Escherichia coli mutant is Escherichia coli BLT40 (accession number: KFCC11603P).

Also provided is a method for producing thymidine using the Escherichia coli mutant strain.

When such an E. coli mutant according to the present invention is used, cyimidine having a purity of 99% or more can be mass-produced at an industrial level.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic view for explaining a process for removing a reusable metabolic pathway enzyme in Example 2 of the present invention;
FIG. 2 is a graph showing the results of performing a thymidine degradation assay on the strains from which the reusable metabolic pathway enzyme has been removed in Example 2 of the present invention;
FIG. 3 is a diagram showing DNA base sequences of udhA (a), NAD kinase ( yfjB ) (b) and pspB (phage shock protein B) (c) expressed according to Example 3 of the present invention,
FIG. 4 is a schematic diagram illustrating a gene insertion process for suppressing the suppression gene and enhancing the reducing power in Example 3 of the present invention. FIG.
5 is a schematic diagram illustrating a vector map for expression of the pyrBIECD operon in Example 4 of the present invention,
6 is a view for explaining a method of selecting a thymidine producing strain in Example 5 of the present invention,
7 is a graph showing the results of HPLC analysis of thymidine produced according to Example 6 of the present invention,
FIG. 8 is a graph showing the results of culturing a 500-L pile lot of mutant strains in Example 7 of the present invention,
9 is a flow chart exemplarily showing a process for purifying thymidine produced using a mutant strain in Example 8 of the present invention,
10 is a graph illustrating the purification of thymidine using Amberlite XAD-16 in Example 8 of the present invention,
11 is a graph comparing HPLC analysis results of standard thymidine (1 g / l, Sigma, A) and purified thymidine (1 g / l, B) in Example 8 of the present invention.

Hereinafter, the present invention will be described in detail with reference to examples. Prior to this, terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary terms, and the inventor should appropriately interpret the concepts of the terms appropriately The present invention should be construed in accordance with the meaning and concept consistent with the technical idea of the present invention. Accordingly, it is to be understood that the constituent features of the embodiments described herein are merely the most preferred embodiments of the present invention, and are not intended to represent all of the inventive concepts of the present invention, so that various equivalents, And the like.

Pyrimidine deoxynucleosides, including thymidine, are synthesized in all proliferating organisms, but since they are strongly regulated and limited, they are produced through low-cost, high-cost organic synthesis to date.

Lee et al. (See Non-Patent Document 2) describe a mechanism of regulating thymidine biosynthetic pathway for isolation of Escherichia coli mutants having thymidine production ability.

Referring to Lee et al., In order to mass-produce thymidine by a biological method using E. coli, the present invention first inhibited the decomposition of thymidine by blocking the reuse pathway in the thymidine biosynthetic pathway. DeoA , udp , upp And tdk were designated as 'BL-T24'. It was confirmed that thymidine degradation assay did not decompose the initial thymidine until 18 hours after the reaction.

In addition, in the present invention, udhA (transhydrogenase) and NADK (NAD kinase) were expressed in order to directly increase cytidine production through direct reinforcement of the cytidine biosynthetic pathway, thereby reinforcing the reducing power inside the cells.

PyrBI (aspartate transcarbamylase), PyrC (dihydroorotase), PyrD (dihydroorotate dehydrogenase) and PyrE (orotate phosphoribosyltransferase), which act on the initiation of thymidine synthesis, were also expressed. These are enzymes regulated by the concentration of nucleotides such as UTP (uridine triphosphate) and CTP (cytidine triphosphate), which are present inside the cell. Therefore, they express additional expression of transcription / expression weakness of the enzyme which can be caused by high concentration of thymidine production . In addition, pbp (phage shock proteins, Psp operon) was expressed together to stabilize the strain.

In addition, accumulation of some nucleotides occurring during the modified biosynthetic pathway can regulate the synthesis of carbamoyl-asapartate, which is the first step of biosynthesis, so that purR , known as a repressor gene involved in the synthesis, The pepA and argR genes were removed to produce a strain designated 'BL-T30'.

On the other hand, the mutant strains according to the present invention in which the biosynthetic regulatory gene and thymidine degrading enzyme were removed showed growth inhibition (6 g / l) in medium containing high concentration of thymidine compared with the parent strain. To improve this, strains resistant to high concentrations of 5'-fluorouracil, hydroxyurea and trimethoprim, resistant to 20g / l thymidine concentration, were selected To give the final strain 'BL-T33'.

The resulting thymidine production strain BL-T33 produced 7 g / l of thymidine through a 7-L fermentation using a final strain BL-T40 in which a specific vector (pTrc99y-pyrBIECD) was inserted And 500 g of pilot in the case of 120 g of 7 g / l of thymidine. Also, a purification process of the produced thymidine was established to finally produce thymidine having a purity of 99%.

Accordingly, the present invention having the nucleotide sequence represented by gene udhA, SEQ ID NO: 2 having the nucleotide sequence to remove the gene deoA, tdk, upp and udp involved in the re-use pathway (salvage pathway) is, described in SEQ ID NO: 1 A gene pspB having a nucleotide sequence represented by SEQ ID NO: 3 and a gene yfjB is expressed, purR , pepA and argR , which are involved in the inhibition of carbamoyl-aspartate synthesis, are removed and a gene pyrBI , pyrC, pyrD and pyrE and starts the ssayimi Dean (thymidine) E. coli mutants have a production capacity over-expression, in accordance with a preferred embodiment of the invention the E. coli mutant Escherichia coli BLT40 (accession number: KFCC11603P).

Hereinafter, the present invention will be described in detail with reference to examples.

Example  1: High concentration Cyimidine  Selection of resistant strains

Prior to the development of thymidine production strains, there was a need to produce resistant strains that did not inhibit growth even at high concentrations of thymidine produced by them. A mutant library was constructed using the mini-transposon mutagenesis method for the screening of thymidine resistant bacteria. A vector (pMV23 transposon vector) was introduced into the E. coli strain of the parent strain to induce a random insertion mutation. Then, resistant bacteria which did not inhibit growth by growth on LB medium containing 10 g / L thymidine were firstly selected and named 'BL-T10'. BL-T10 was UV-mutated to select resistant bacteria that did not show growth inhibition even at 20 g / l thymidine medium, which was named 'BL-T20'.

Example  2: Cyimidine  Reusable metabolic pathways for production ( salvage pathway ) Blocking 티미딘 phosphorylase ( deoA ), 티미딘 kinase ( tdk ), UMP  pyrophosphorylase ( upp ) And uridine phosphorylase ( udp ) gene)

DeoA , udp, and upp involved in the reuse pathway are genes that express enzymes that attach or hydrolyze (deoxy) ribose (deoxyribose) to the nucleobase. The degradation of thymidine The genes were removed. In addition, thymidine kinase (tdk) that binds phosphate to thymidine was removed to block the conversion to dTMP.

In order to knock out each of the above genes, a PCR fragment having a sequence complementary to 50 nt at both ends of the gene was used as shown in Fig. This fragment contains the chloramphenicol gene, which is a selection marker, and contains deoA (Accession No. AAC75351), tdk (accession No. AAC74320), upp (accession No. ACB03650) and udp (accession No. ACB04853) And a 50 nt sequence at both ends. When this fragment is transformed into a host, it is homologous recombined with the end of each gene in the genomic DNA by the FLP recombinase. The target gene is removed and the chloramphenicol gene in place can be removed by expressing the FLP helper plasmid. The strain in which all the genes were removed was named 'BL-T24' as a mutation that lost the cytidine-resolving ability.

In this way, thymidine degradation assay was performed with the 'BL-T24' strain in which the reuse metabolic pathway enzyme was removed and the 'BL21' strain in the parent strain to confirm that thymidine degradation pathway was blocked. The results are shown in Fig. Referring to FIG. 2, in the case of the parent strain 'BL21', about 40% of thymidine was decreased after 1 hour of reaction, and almost all thymidine was degraded after 4 hours. However, in case of BL-T24, It can be confirmed that the initial thymidine did not decompose and the decomposition of thymidine by the reuse metabolic pathway was blocked.

Example  3: inhibition ( repressor ) gene( purR , pepA  And argR ) And the foreign gene udhA , yfjB  And pspB ) Expression

The level of NADPH in cytidine production is already known to be an important part (HC Lee, JS Kim, W. Jang, SY Kim, 2010, High NADPH / NADP + ratio improves thymidine production by ametabolically engineered strain, J. Biotechnol. 20, 149 (1-2): 24-32; H. Fang, W. Xie, Q. Xu, C. Zhang, N. Chen, 2013, Enhancement of cytidine production by coexpression of gnd , zwf , and prs genes in recombinant Escherichia coli CYT15., Biotechnol. Lett., 35 (2): 245). In order to increase the intracellular concentration of NADPH, there is a method of overexpressing a related gene promoting the conversion of NADP to NADPH. Related genes include membrane-bound transhydrogenase, pntAB, and soluble transhydrogenase, udhA (KA Datsenko, BL Wanner, 2000, One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR product., PNAS., 97 (12): 6640-6645). In addition, since NADP, which is a precursor of NADPH, is mainly present in the form of NADPH in the cells, strengthening of NAD kinase that converts NAD to NADP may lead to increase of NADPH. In the present invention, as shown in FIG. 3, overexpression of soluble hydrogen transport enzymes udhA (a) and NAD kinase ( yfjB ) (b) enhances the internal reducing ability of the cells. To stabilize cells that can become unstable, pspB (phage shock protein B) (c).

The expression of udhA , NADK and pspB for the removal of the inhibitory gene and for the enhancement of the reducing power was performed using the above-described complementary PCR fragment as shown in FIG. First, a plasmid pCM1 was prepared by cloning pKD3's chloramphenicol and FRT (FLP recognition target) into a vector having an rrnB promoter / terminator (pBAD vector).

Soluble hydrogen transferase gene obtained from E. coli (udhA), was prepared E. coli NAD kinase gene (yfjB) and by cloning the gene on the pspB pCM1 pCM- udhA, pCM-NADK pCM- pspB and, each primer was used (primer ) Are shown in Table 1 below.

Accumulation of some nucleotides occurring during the modified biosynthetic metabolism of the mutant strain according to the present invention may lead to the inhibition of carbamoyl-aspartate synthesis, which is the first step of biosynthesis. This suppression and gene purR, pepA and argR known as participating in, to remove these genes have been identified, more than three times the carA enzyme increases, which has been reported to lead to an increase in ssayimi Dean production (BS Koo, HH Hyun , SY Kim, CH Kim, HC Lee, 2011, Enhancement of thymidine production in E. coli by elimination repressors regulating the carbamoyl phosphate synthetase operon., Biotechnol. Lett. 33 (1): 71-78). Therefore, the three inhibitory genes were removed in order to enhance the production of thymidine by enhancing the step of introducing thymidine biosynthesis. In order to insert the expression cassette into the expression cassette, Section.

to the pCM- udhA as the template for PCR was udhA expression cassette fragment (fragment expression cassette) containing purR both ends and 50nt complementary sequence, and a pCM-NADK as a template pepA both ends NADK containing 50nt complementary to the sequence The expression cassette fragment was PCR, and a pspB expression cassette fragment containing a sequence complementary to 50 nt of both ends of argR was used as a template for pCM- pspB . The primer sequences used are shown in Table 2 below.

Each fragment was transformed into a host and homologous to the end of each gene in genomic DNA by FLP recombinase. The target gene was removed and the chloramphenicol gene in the place was removed by expressing the FLP helper plasmid, and the inserted udhA , yfjB and pspB were inserted into the genomic DNA (see Fig. 4) .

Example  4: Cyimidine  Expression of Biosynthetic Pathway Gene aspartate  transcarbamylase ( PyrBI ), 디 디오 로로토스 ( PyrC ), 이 디오 로 오테이트  dehydrogenase ( PyrD ) And orotate phosphoribosyltransferase ( PyrE ))

In the pyrimidine biosynthetic phase, the initial conversion of aspartate to uridine monophosphate (UMP) is carried out by pyr operon. These enzymes do not depend on specific transcription factors, (L. Charles, J. Turnbough, RL Switzer, 2008, Regulation of pyrimidine biosynthetic gene expression in bacteria: repression without repressors, Microbiol. Mol. Biol Rev., 72 (2): 266-300). The developed strain according to the present invention produced through the manipulation of the metabolic pathway can cause the accumulation of specific nucleotides nonspecifically than normal cells, and the suppression of the inhibition of these enzymes is expected.

Therefore, in the present invention, pyrBI , pyrC , pyrD and pyrE genes corresponding to pyr operon are inserted into a vector (pTrc99y vector) as shown in FIG. 5 so as to avoid the regulatory mechanism through continuous expression of these enzymes and increase the production of thymidine Lt; / RTI > A yeast recombination system was used to construct a plasmid expressing the pyr operon. Four PCR fragments were inserted into pTrc99y to construct pTrc99y-pyrBIECD. Each PCR fragment was obtained using the primers shown in Table 3 below.

Example  5: Nucleoside  Analog ( nucleoside analogues ) Resistant strains

BL-T30 ', a mutant strain in which the biosynthetic regulatory gene and thymidine degrading enzyme are removed, shows growth inhibition (6 g / l) in medium containing high concentration of thymidine. To improve this, strains resistant to high concentrations of 5-fluorouracil, hydroxyurea, and trimethoprim at a concentration of 20 g / l thymidine were cultured in a stepwise manner And selected as the final 'BL-T33'.

First, 'UV-mutation' was performed on 'BL-T30' and strains growing on medium supplemented with pyrimidine analogue 5-fluorouracil were selected and named 'BL-T31'. The strains showing resistance in 100 mM 5-fluoro uracil can be assayed for cytidine production ability in the complete minimal media with the composition shown in Table 4 below using the screening system described in Fig.

Then, UV mutation was performed again on 'BL-T31', and a hydroxyurea resistant strain was selected and named 'BL-T32'. Finally, UV-mutation was performed on 'BL-T32', and then the strain-resistant strain was selected and named 'BL-T33'. 'BL-T33' was resistant to both 5-fluorouracil, hydroxyurea and trimethoprim. BL-T40 was constructed by inserting pTrc99y-pyrBIECD vector into this strain. The mutant BL- T40 'on Dec. 12, 2014 to the Korean Culture Center of Microorganisms (KCCM) and received the accession number KFCC11603P.

The thymidine production ability of the stepwise strains ranging from 'BL-T30' to 'BL-T40' was measured and shown in the following Table 5, and it can be seen that the mutation progressively increased as the mutation progressed. The final strain BL-T40 showed a high production rate of 1,649 mg / L when cultured on a flask scale with thymidine production medium.

Example  6: Cyimidine  7ℓ for production Oil price formula  culture

In order to confirm the cytidine productivity of the 'BL-T40' strain in a 7-liter fermentor, the cultivation was carried out by fed-batch culture. The composition of the production medium for producing thymidine is shown in Table 6 below.

Thymidine production strain 'BL-T40' was inoculated in 5 ml of LB medium and pre-cultured at 37 ° C and 200 rpm. Then, 1 ml and 1.5 ml of the preculture were inoculated into a 500 ml baffled flask containing 100 ml and 150 ml of LB medium and cultured at 37 ° C and 200 rpm. When 3OD was reached, the cells were inoculated on a 7 L jar fermenter containing 2.5 L of the production medium and cultured at 34 ° C., 500 rpm, and 1 vvm. The pH was adjusted to maintain the pH at 7.0 using ammonium hydroxide (NH ₄ OH), and the concentration of glycerol, a carbon source during the culture, was measured in real time. When the concentration of glycerol in the medium fell below 30 g / . At the end of the 90-hour carbon source consumption, the final concentration of thymidine was found to be 7.1 g / l. The amount of thymidine was quantitatively determined by a method in which the concentration of the medium was determined by HPLC .

Example  7: Cyimidine  500ℓ for production Pilots  culture

Production of thymidine in a 500 l pilot lot was carried out using the indicated medium in the 7 liter culture described above. The first seed (1 ^st seed) was prepared by inoculating 'BL-T40' into a 500 ml baffle flask containing 50 ml of LB medium and inoculating 50 ml of primary seed solution into a 7 l fermenter containing 4 l of seed medium after 12 hours incubation with 37 ℃, 500rpm, conditions of 1.0vvm to prepare a secondary seed (seed 2 ^nd). Then, 8 liters of the secondary seed liquid obtained through two 7-liter jar cultures were inoculated in a 500-L pile containing 300 L production medium and cultured at 34 ° C. and 260 to 480 rpm for 120 hours to produce thymidine. During the culture, the aeration amount and the internal pressure were adjusted in the range of 0.5 to 1.0 vvm and 0.1 to 0.5 kg / cm 3. The concentration of glycerol, which is a carbon source, during the culture was analyzed to prevent the sugar content from dropping below 30 g / L, and the feeding amount was adjusted to maintain the sugar concentration of the culture medium at 60 to 80 g / L.

As shown in FIG. 8, the production of thymidine exhibited mass production of a typical primary metabolite in which the production concentration increased with the growth of bacteria at the initial stage of culture, and at the time of consumption of 90 hours of culture The bacterial growth reached the stationary phase and the production rate of thymidine gradually decreased. The cell concentration of the final fermentation broth was 140 OD and the concentration of thymidine produced was 7.6 g / ℓ for 120 hours.

Example  8: Cyimidine  Purification process

9 is a flowchart exemplarily showing a purification process of thymidine produced using the mutant strain of the present invention. After the culture was completed, the cells were removed and the resulting culture was subjected to two steps of a micro-filtration system with a pore size of 1 to 10 쨉 m to remove impurities. Thereafter, the solution was concentrated to a concentration of about 30 g / l of thymidine using a nano-filter or a vacuum-evaporator. Then, activated carbon A (without thymidine binding) was added thereto in a volume ratio of 3% (w / v), followed by shaking for 1 hour to bind components other than thymidine . The activated carbon was then filtered to obtain an activated carbon A-free aqueous solution. Thymidine was added to the solution containing 3% (w / w) of activated carbon B to bind thymidine. Then, the solution removed by vacuum filtering is removed, and a cakes of thymidine-activated carbon (B) remaining is washed in a bead volume of 3 times, Respectively. The elution of the final thymidine was performed with 20% isopropyl alcohol (see Table 7 and Figure 10).

The fraction obtained after the elution showed a cytidine concentration of about 80 g / l, and then crystallization was carried out at 4 캜. Crystallized thymidine was obtained by filtration and then slowly washed with 4 ° C deionized water. In order to obtain the maximum yield, the recrystallization process was repeated for the thymidine dissolved in the wash. The thymidine crystals were dried in an oven at 50 < 0 > C for 12 hours and HPLC analysis was performed to determine the purity of thymidine. As shown in FIG. 11, it was confirmed that cyimidine having a purity of 99% can be obtained through the above purification process as a result of comparison with standard thymidine (Sigma).

While the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, Such modifications and changes are to be considered as falling within the scope of the following claims.

Korea Microorganism Conservation Center (Domestic) KFCC11603P 20141212

<110> ForBioKorea Co., Ltd. <120> Fermentation process for preparing thymidine by the recombinant          E. coli <130> NP14-12224 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 1401 <212> DNA <213> Artificial Sequence <220> <223> E. coli soluble transhydrogenase (udhA) <400> 1 atgccacatt cctacgatta cgatgccata gtaataggtt ccggccccgg cggcgaaggc 60 gctgcaatgg gcctggttaa gcaaggtgcg cgcgtcgcag ttatcgagcg ttatcaaaat 120 gttggcggcg gttgcaccca ctggggcacc atcccgtcga aagctctccg tcacgccgtc 180 agccgcatta tagaattcaa tcaaaaccca ctttacagcg accattcccg actgctccgc 240 tcttcttttg ccgatatcct taaccatgcc gataacgtga ttaatcaaca aacgcgcatg 300 cgtcagggat tttacgaacg taatcactgt gaaatattgc agggaaacgc tcgctttgtt 360 gacgagcata cgttggcgct ggattgcccg gacggcagcg ttgaaacact aaccgctgaa 420 aaatttgtta ttgcctgcgg ctctcgtcca tatcatccaa cagatgttga tttcacccat 480 ccacgcattt acgacagcga ctcaattctc agcatgcacc acgaaccgcg ccatgtactt 540 atctatggtg ctggagtgat cggctgtgaa tatgcgtcga tcttccgcgg tatggatgta 600 aaagtggatc tgatcaacac ccgcgatcgc ctgctggcat ttctcgatca agagatgtca 660 gattctctct cctatcactt ctggaacagt ggcgtagtga ttcgtcacaa cgaagagtac 720 gagaagatcg aaggctgtga cgatggtgtg atcatgcatc tgaagtcggg taaaaaactg 780 aaagctgact gcctgctcta tgccaacggt cgcaccggta ataccgattc gctggcgtta 840 cagaacattg ggctagaaac tgacagccgc ggacagctga aggtcaacag catgtatcag 900 accgcacagc cacacgttta cgcggtgggc gacgtgattg gttatccgag cctggcgtcg 960 gcggcctatg accaggggcg cattgccgcg caggcgctgg taaaaggcga agccaccgca 1020 catctgattg aagatatccc taccggtatt tacaccatcc cggaaatcag ctctgtgggc 1080 aaaaccgaac agcagctgac cgcaatgaaa gtgccatatg aagtgggccg cgcccagttt 1140 aaacatctgg cacgcgcaca aatcgtcggc atgaacgtgg gcacgctgaa aattttgttc 1200 catcgggaaa caaaagagat tctgggtatt cactgctttg gcgagcgcgc tgccgaaatt 1260 attcatatcg gtcaggcgat tatggaacag aaaggtggcg gcaacactat tgagtacttc 1320 gtcaacacca cctttaacta cccgacgatg gcggaagcct atcgggtagc tgcgttaaac 1380 ggtttaaacc gcctgtttta a 1401 <210> 2 <211> 879 <212> DNA <213> Artificial Sequence <220> <223> E. coli NAD kinase (yfjB) <400> 2 atgaataatc atttcaagtg tattggcatt gtgggacacc cacggcaccc cactgcactg 60 acaacacatg aaatgctcta ccgctggctg tgcacaaaag gttacgaggt catcgttgag 120 caacaaatcg ctcacgaact gcaactgaag aatgtgaaaa ctggcacgct cgcggagatt 180 gggcaactag ctgatctcgc ggtagtcgtt ggtggcgacg gtaatatgct gggcgcggca 240 cgcacactcg cccgttacga tattaaagtt attggaatca accgtggcaa cctgggtttc 300 ctgactgacc ttgaccccga taacgcccag caacagttag ccgatgtgct ggaaggccac 360 tacatcagcg agaaacgttt tttgctggaa gcgcaagtct gtcagcaaga ttgccagaaa 420 cgcatcagca ccgcgataaa tgaagtggtg cttcatccag gcaaagtggc gcatatgatt 480 gagttcgaag tgtatatcga cgagatcttt gcgttttctc agcgatctga tggactaatt 540 atttcgacgc caacaggctc caccgcctat tccctctctg caggcggtcc tattctgacc 600 ccctctctgg atgcgattac cctggtgccc atgttcccgc atacgttgtc agcacgacca 660 ctggtcataa acagcagcag cacgatccgt ctgcgttttt cgcatcgccg taacgacctg 720 gaaatcagtt gcgacagcca gatagcactg ccgattcagg aaggtgaaga tgtcctgatt 780 cgtcgctgtg attaccatct gaatctgatt catccgaaag attacagtta tttcaacaca 840 ttaagcacca agctcggctg gtcaaaaaaa ttattctaa 879 <210> 3 <211> 225 <212> DNA <213> Artificial Sequence <220> E. coli phage shock protein B (pspB) <400> 3 atgagcgcgc tatttctggc tattccgtta accatttttg tgctgtttgt tttaccgatc 60 tggttatggc tgcattacag caatcgttct ggtcgcagtg aattgtcgca aagtgagcag 120 cagcgattag cgcaactggc tgatgaagca aaacggatgc gcgaacgtat tcaggcgctg 180 gaatctattc ttgatgcaga acatccgaac tggagggatc gctaa 225

Claims (3)

The re-pathway genes deoA, tdk, upp and udp involved in (salvage pathway) is removed, and gene yfjB and a sequence consisting of the nucleotide sequence represented by gene udhA, SEQ ID NO: 2 consisting of the nucleotide sequence represented by SEQ ID NO: 1 The gene pspB consisting of the nucleotide sequence of SEQ ID NO: 3 is expressed and the genes purR , pepA and argR involved in the inhibition of carbamoyl-aspartate synthesis are removed and the genes pyrBI , pyrC and pyrD constituting the pyr operon and pyrE overexpression ssayimi Dean (thymidine) E. coli mutants have a production capacity. The method according to claim 1,
Wherein the Escherichia coli mutant strain is Escherichia coli BLT40 deposited in the Korean Microorganism Conservation Center under the deposit number KFCC11603P. A method for producing thymidine using the Escherichia coli mutant of claim 1 or 2.

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