KR101807775B1 - Producing method of cadaverin by recycling of pyridoxal-5-phosphate with pyridoxal kinase - Google Patents

Abstract

The present invention relates to a method of producing cadaverine by reusing pyridoxal phosphate after the application of pyridoxal kinase. More specifically, a recombinational strain, which simultaneously expresses pyridoxal kinase (pdxY) and lysine decarboxylase (cadA) derived from Escherichia coli, is used and pyridoxal (PL) or pyridoxine (PN) is reused as pyridoxal-5-phosphate, and thus, the mass production of cadaverine is enabled at a low cost through a simple process.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing cadaverine by re-use of pyridoxal phosphate by applying pyridoxal kinase to pyridoxal-5-phosphate with pyridoxal kinase,

The present invention, more particularly, to the E. coli on the cadaverine production method through the re-use of pyridoxal phosphate by applying the pyridoxal kinase (Escherichia coli) lysine decarboxylase derived from (cadA, lysine decarboxylase) and pyridoxal kinase (pdxY, pyridoxal using the recombinant strain expressing the pyridoxal kinase) at the same time (pyridoxal, PL) or pyridoxine (pyridoxine, PN) of (Pyridoxal-5-phosphate), thereby producing a large amount of cadaverine in a low cost and simple process.

Development of industrial strains for the production of bio-based plastics produced for the purpose of replacing petroleum-derived plastics have been studied extensively. In this regard, nylon monomers such as cadverine, putrescine are used in Escherichia coli ) or Corynebacterium ( Corynebacterium < RTI ID = 0.0 > glutamicum ) have been studied by fermentation from glucose or xylose (Korean Patent No. 10-1231897; European Patent Publication No. 2540836; Qian ZG, et. al., Biotechnol Bioeng . 2009 Nov 1; 104 (4): 651-62; Qian ZG, et al., Biotechnol Bioeng . 2011 Jan; 108 (1): 93-103). However, when such a method is used, productivity and concentration are relatively inefficient and thus it has been difficult to utilize it industrially.

Recently, in order to overcome this problem, a case has been reported in which a high concentration of cadaverine is produced by a whole-cell reaction from lysine (L-lysine), which is a mass-produced precursor in several companies (Ma W, et al., Biotechnol Lett . 2015 Apr; 37 (4): 799-806). Compared with the fermentation process, the whole-cell reaction can deliver a high concentration of reaction in a short period of time, and it is possible to avoid a variety of by-products during the fermentation, have.

For this reaction, lysine dicarboxylase is used. In this case, pyridoxal phosphate (pyridoxal phosphate, PLP) is required to enter the reaction, and as the reaction proceeds, PLP reacts with pyridoxal pyridoxal, PL), which can be converted back to PLP by intracellular ATP and used for the reaction. The circulation process of PLP is usually time-consuming and leads to a phenomenon that conversion rate is decreased due to lack of PLP when high-concentration whole cell reaction proceeds. In order to overcome this drawback, recently, in the method of producing cadaverine using whole cell culture, 0.1 mM of external PLP is added together with lysine as a substrate in the reaction environment. However, since PLP is very expensive, .

In the biosynthesis process using enzymes, various enzymes require coenzyme. In addition to PLP, such coenzymes have various substances such as NADPH, NADP, ATP, sugar nucleotide and PAPS. However, most of these coenzyme substances are expensive and unstable, and thus the structure is liable to be broken. Thus, a coenzyme reprocessing system is proposed for effective application in a process using enzymes requiring coenzyme. This reproductive system of the coenzyme can be solved by using an enzyme-enzyme or enzyme-coupling reaction (Zhao, Huimin, and Wilfred A. van der Donk. Current Opinion in Biotechnology 14.6 (2003): 583 -589.). Enzyme-enzyme coupling reactions are most commonly used for the reproduction of coenzyme, and two enzymes acting in pairs can work together to reproduce the coenzyme.

PLP is converted to PL as a coenzyme in the reaction of lysine dicarboxylase, suggesting that pyridoxal kinase can be used as an enzyme capable of converting PL to PLP again. Among them, the pyridoxal kinase pdxY or pdxK derived from E. coli K-12 has been reported (Yang, Yong, et al., Journal of bacteriology 180.7 (1998): 1814-1821). However, there has been no report on the use of pdxY in the production of the cataractine or in the reproduction of PLP.

Accordingly, the present inventors have made efforts to develop a more economical and efficient cadaverine production system through recycling (reuse) of PLP coenzyme. As a result, it has been found that lysine decarboxylase (cadA) derived from Escherichia coli and pyridoxal kinase , pdxY) were synthesized. When recombinant E. coli was transformed into cadalin by cadA, and pdxY was converted into PL, pdxY converted PL to PLP and could be used again in cadA The PLP reuse system can be used. In addition, through the PLP reuse system of the present invention, it has been confirmed that PLP can be effectively reused in a reaction solution in which PL or PN as a coenzyme is added at a relatively low cost, thereby converting lysine into cadaverine.

Thus, the present inventors have found that a recombinant Escherichia coli that simultaneously expresses lysine decarboxylase (cadA) and pyridoxal kinase (pdxY) derived from Escherichia coli can be used to produce a cadaverine production process that effectively reuses PLP coenzyme Thereby completing the present invention.

Accordingly, it is an object of the present invention to provide an economical and efficient method for producing cadaverine by reusing PLP by applying pdxY.

In order to achieve the above object,

i) preparing a recombinant microorganism that simultaneously expresses a lysine decarboxylase and a pyridoxal kinase;

ii) culturing and obtaining the recombinant microorganism produced in step i);

iii) suspending and reacting the recombinant microorganism obtained in step ii) in a reaction solution containing a lysine substrate and a coenzyme of lysine decarboxylase; And

iv) obtaining the cadaverine from the reaction solution reacted in step iii) above.

The present invention also provides a method for producing a recombinant microorganism, comprising the steps of: i) preparing a recombinant microorganism expressing lysine decarboxylase and pyridoxal kinase simultaneously;

ii) inoculating and culturing the recombinant microorganism prepared in step i) in a medium containing a lysine substrate and a coenzyme of lysine decarboxylase;

iii) obtaining the cadaverine from the medium cultured in the step ii).

In a preferred embodiment of the present invention, the lysine decarboxylase of step i) may be a cadA derived from E. coli consisting of the amino acid sequence of SEQ ID NO: 2.

In another preferred embodiment of the present invention, the pyridoxal kinase of step i) may be pdxY derived from E. coli consisting of the amino acid sequence of SEQ ID NO: 4.

In another preferred embodiment, the microorganism of the step i) of the present invention may be any one of Escherichia coli (E. coli), Corynebacterium (Corynebacterium) or hafnia (Hafnia).

In another preferred embodiment of the present invention, the coenzyme of the lysine decarboxylase is selected from the group consisting of pyridoxal phosphate (Pyridoxal Phosphate, PLP), pyridoxal (PL) and pyridoxine (PN ). ≪ / RTI >

In another preferred embodiment of the present invention, the mass production of the cadaverine may be carried out under aerobic conditions and at a temperature of 20 to 40 DEG C for 2 to 5 days.

Accordingly, the present invention provides an economical and efficient method for producing cadaverine by reusing PLP by applying pdxY.

The recombinant Escherichia coli expressing the lysine decarboxylase (cadA) and the pyridoxal kinase (pdxY) simultaneously produced from Escherichia coli according to the present invention can be effectively used for PLP recycling and production of cadaverine . Specifically, when cadA converts lysine to cadaverine, when PLP is converted to PL, pdxY can convert PL to PLP and be used again in cadA, and mass production of cadaverine capable of reusing PLP ≪ / RTI >

The PLP reuse system according to the present invention can effectively reuse the PLP even if a relatively low cost PL or PN is used as the coenzyme without using the expensive PLP required in the cataract production process through the conventional biological method The lysine can be converted into cadaverine, so that the mass production method of the cadaverine of the present invention is economical and efficient.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a conceptual diagram of PLP regeneration and a conceptual diagram of PLP precursors and enzymes utilized in the present invention. FIG.
FIG. 2 shows a vector map of each plasmid construct for overexpressing lysine decarboxylase (cadA) and pyridoxal kinase (pdxY) derived from Escherichia coli.
Figure 3 shows SDS-PAGE results to confirm the expression and size of cadA and pdxY expressed in recombinant E. coli.
Figure 4 shows confirmation of production of cadaverine using recombinant E. coli overexpressing cadA and pdxY.
Fig. 5 shows the effect of producing cadaverine depending on the type of coenzyme added to the reaction solution, using recombinant E. coli overexpressing cadA and pdxY:
5A shows the production effect of cadaverine produced in a reaction solution to which PLP is added as a coenzyme;
FIG. 5B shows the production effect of cadaverine produced in a reaction solution to which no coenzyme is added;
Fig. 5C shows the production effect of cadaverine produced in the reaction solution to which PL is added as a coenzyme; And
Fig. 5D shows the production effect of cadaverine produced in a reaction solution to which PN is added as a coenzyme.

Hereinafter, the present invention will be described in detail.

As described above, PLP is indispensably required as a coenzyme of lysine decarboxylase in the production process of cadaverine using the bioconversion reaction. However, since the PLP is expensive, conventional production process of cadaverine has not been economically feasible. However, no economical method of producing cadaverine using a PLP recycling system has been reported.

The method of producing cadaverine according to the present invention uses recombinant Escherichia coli expressing both lysine decarboxylase (cadA) and pyridoxal kinase (pdxY) derived from Escherichia coli, Can be reused, making economical mass production of cadaverine possible.

Therefore,

i) preparing a recombinant microorganism that simultaneously expresses a lysine decarboxylase and a pyridoxal kinase;

ii) culturing and obtaining the recombinant microorganism produced in step i);

iii) suspending and reacting the recombinant microorganism obtained in step ii) in a reaction solution containing a lysine substrate and a coenzyme of lysine decarboxylase; And

iv) obtaining the cadaverine from the reaction solution reacted in step iii) above.

The " lysine dicarboxylase " in step i) of the present invention may be any enzyme known in the art which is capable of converting lysine into cadaverine. Specifically, the lysine decarboxylase is preferably derived from Escherichia coli. More specifically, the lysine decarboxylase is preferably cadA consisting of the amino acid sequence of SEQ ID NO: 2, but is not limited thereto. The nucleotide sequence of SEQ ID NO: A protein encoded by a nucleotide sequence in which one or more genes are added, deleted or substituted can be used.

The "pyridoxal kinase" of step i) of the present invention may be any enzyme known in the art which is capable of converting PL or PN into PLP. More specifically, it is preferable that the pyridoxal kinase is derived from E. coli. More specifically, the pyridoxal kinase is preferably pdxY consisting of the amino acid sequence of SEQ ID NO: 4, but is not limited thereto. The nucleotide sequence of SEQ ID NO: 3, A protein encoded by a base sequence in which two or more genes are added, deleted, or substituted can also be used. PdxK derived from Escherichia coli can also be used as a pyridoxal kinase similar to the above pdxY. However, when pdxK is used in the production process of cadaverine, effective PLP reuse is not exhibited and the effect of producing cadaverine is low and compared with using pdxY under the same conditions It is not effective.

The "microorganism" of step i) of the present invention may be any gene known in the art that can be transformed and recombined with a plasmid constructed so as to overexpress a foreign gene. Specifically, the microorganism is preferably any one of E. coli , Corynebacterium , or Hafnia . More preferably, the microorganism is E. coli, but is not limited thereto.

The " coenzyme of lysine dicarboxylase " of step iii) of the present invention is composed of pyridoxal phosphate (Pyridoxal Phosphate, PLP), pyridoxal (PL) and pyridoxine , And the like. However, since PLP is expensive, it is more preferable to use PL or PN with a relatively low price. When PL or PN is used alone with a small amount of PLP, the activity of lysine decarboxylase can be exhibited from the beginning of the reaction, so it is not limited thereto.

The " reaction " of the step iii) of the present invention is preferably carried out under aerobic conditions and at a temperature of 20 to 40 DEG C for 2 to 5 days, more specifically in aerobic conditions and at a temperature of 30 to 40 DEG C for 24 to 72 hours But is not limited thereto.

In a specific example of the present invention, we prepared recombinant E. coli overexpressing lysine decarboxylase (cadA) and pyridoxal kinase (pdxY) (FIGS. 2 and 3). As a result of the bioconversion reaction to convert lysine into cadaverine using the recombinant E. coli thus prepared, the experimental group expressing cadA and pdxY as compared with the negative control group using E. coli expressing only cadA showed not only PLP but also PL or PN (Fig. 5), the conversion of lysine to cadaverine in the reaction solution added as a coenzyme showed an effective production amount (Fig. 5).

In addition, pdxK, known as a pyridoxal kinase derived from Escherichia coli, in association with pyridoxal kinase, did not exhibit effective cadavagin production in all cases using PLP or PL as a coenzyme even when coexpressed with cadA, but expressed pdxY The PL was effectively converted into PLP in the experimental group, and PLP could be recycled to convert lysine to cadaverine (Fig. 4).

Therefore, through the PLP reusing system according to the present invention, it is possible to reuse the PLP even if the relatively low cost PL or PN is used as the coenzyme without using the expensive PLP required in the cadaverine production process by the conventional biological method Can effectively take place and convert the lysine to cadaverine, the mass production method of the cadaverine of the present invention is economical and efficient.

In addition,

i) preparing a recombinant microorganism which simultaneously expresses lysine decarboxylase and pyridoxal kinase;

ii) inoculating and culturing the recombinant microorganism prepared in step i) in a medium containing a lysine substrate and a coenzyme of lysine decarboxylase;

iii) obtaining the cadaverine from the medium cultured in the step ii).

The " lysine dicarboxylase " in step i) of the present invention may be any enzyme known in the art which is capable of converting lysine into cadaverine. Specifically, the lysine decarboxylase is preferably derived from Escherichia coli. More specifically, the lysine decarboxylase is preferably cadA consisting of the amino acid sequence of SEQ ID NO: 2, but is not limited thereto. The nucleotide sequence of SEQ ID NO: A protein encoded by a nucleotide sequence in which one or more genes are added, deleted or substituted can be used.

The "pyridoxal kinase" of step i) of the present invention may be any enzyme known in the art which is capable of converting PL or PN into PLP. More specifically, it is preferable that the pyridoxal kinase is derived from E. coli. More specifically, the pyridoxal kinase is preferably pdxY consisting of the amino acid sequence of SEQ ID NO: 4, but is not limited thereto. The nucleotide sequence of SEQ ID NO: 3, A protein encoded by a base sequence in which two or more genes are added, deleted, or substituted can also be used. PdxK derived from Escherichia coli can also be used as a pyridoxal kinase similar to the above pdxY. However, when pdxK is used in the production process of cadaverine, effective PLP reuse is not exhibited and the effect of producing cadaverine is low and compared with using pdxY under the same conditions It is not effective.

The "microorganism" of step i) of the present invention may be any gene known in the art that can be transformed and recombined with a plasmid constructed so as to overexpress a foreign gene. Specifically, the microorganism is preferably any one of E. coli , Corynebacterium , or Hafnia . More preferably, the microorganism is E. coli, but is not limited thereto.

The " coenzyme of lysine decarboxylase " of step ii) of the present invention is a coenzyme selected from the group consisting of pyridoxal phosphate (PLP), pyridoxal (PL) and pyridoxine , And the like. However, since PLP is expensive, it is more preferable to use PL or PN with a relatively low price. When PL or PN is used alone with a small amount of PLP, the activity of lysine decarboxylase can be exhibited from the beginning of the reaction, so it is not limited thereto.

The "cultivation" of step ii) of the present invention is preferably carried out under aerobic conditions and at a temperature of 20 to 40 ° C for 2 to 5 days, more specifically in aerobic conditions and at a temperature of 30 to 40 ° C for 24 to 72 hours But is not limited thereto.

The "mass production method of cadaverine" of the present invention is a method known in the art so as to produce cadaverine in a single step by culturing the recombinant microorganism through bacth or continuous process, May also be performed.

The PLP reuse system according to the present invention can effectively reuse the PLP even if a relatively low cost PL or PN is used as the coenzyme without using the expensive PLP required in the cataract production process through the conventional biological method The lysine can be converted into cadaverine, so that the mass production method of the cadaverine of the present invention is economical and efficient.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Lysine  Dicarboxylase (lysine decarboxylase ) And pyridoxal kinase kinase , pdxY ) Production of Overexpressed Recombinant Escherichia coli

≪ 1-1 > Preparation of transformant

Lecine dicarboxylase (cadA), an enzyme that produces cadaverine from lysine, and Echerichia coli , which overexpresses the pyridoxal kinase (pdxY) of the present invention, were prepared (FIG. 2).

Specifically, the genome was isolated by culturing E. coli K12 MG1655, and then the cadA gene having the nucleotide sequence of SEQ ID NO: 1 was amplified. Then, the amplified cadA gene was isolated and purified, and inserted into the BamHI and HindIII sites of the pET-24ma plasmid to construct a cadA expression plasmid.

Further, the pdxY gene having the nucleotide sequence of SEQ ID NO: 2 was amplified, separated and purified and inserted into the BamHI and HindIII sites of the pET-23a plasmid using the genome isolated from E. coli K12 MG165 as a template to construct a pdxY expression plasmid Respectively. In order to be used as a comparative control, a plasmid expressing the pyridoxal kinase pdxK derived from E. coli K12 was constructed (Yang, Yong, Genshi Zhao, and Malcolm E. Winkler. FEMS microbiology letters 141.1 (1996) 95.). Then, the constructed cadA expression plasmid and pdxY expression plasmid were simultaneously transformed into E. coli BL21 (lambda DE3) to prepare recombinant Escherichia coli expressing cadA and pdxY.

<1-2> In recombinant E. coli cadA  And pdxY Confirmation of expression

it was confirmed that the recombinant Escherichia coli capable of overexpressing the cadA and pdxY enzymes could effectively express the enzyme through IPTG induction.

Specifically, recombinant E. coli transformed in Example <1-1> was cultured on an LB agar plate for 3 minutes at 37 ° C. for 24 hours to obtain a single colony, broth), incubated at 37 ° C with shaking at 200 rpm, and pre-cultured for 24 hours. Then, 0.5 ml of the culture medium pre-incubated with the cells was inoculated into 50 ml of LB liquid medium and cultured at 25 or 30 ° C with shaking at 200 rpm. Three hours after the initiation of the present culture, 5 μl of 0.1 M IPTG was added to the medium to induce overexpression of cadA and pdxY proteins, and the cultivation was carried out for a total of 24 hours. After culturing, 1 ml of the culture medium was obtained, centrifuged at 13000 rpm, treated with 100 μl of Bugbuster (Novagen), and reacted at 37 ° C for 1 hour to dissolve the cells. After centrifuging the cell lysate at 13000 rpm, the supernatant was obtained as a water-soluble protein layer, mixed with 4 μl of 5 × SDS-PAGE loading buffer, and boiled at 100 ° C. for 5 minutes for sample pretreatment. The pretreated samples were loaded on a 12% SDS-PAGE gel, developed by flowing 120 V, 700 mA current for 2 hours, and then SDS-PAGE gel was stained with a staining solution (0.1% Coomassie Brilliant Blue R-250, 50% methanol And 10% glacial acetic acid for 1 hour.The stained SDS-PAGE gel was decolorized in decolorization solution (50% methanol and 10% glacial acetic acid solution) for 2 hours to identify the resulting protein bands.

As a result, as shown in FIG. 3, the recombinant protein was overexpressed in E. coli according to IPTG induction, and a cadA protein having a molecular weight of about 80 kDa and a pdxY protein band having a molecular weight of about 31 kDa were identified through SDS-PAGE (FIG. 3) . In the case of the pdxY protein, it was confirmed that the protein band appeared when the whole protein was loaded rather than when the cell lysate was centrifuged and only the supernatant was loaded. Therefore, it was confirmed that pdxY exists in the insoluble protein layer.

cadA  And pdxY  Mass production of cadaverine using over-expressed recombinant Escherichia coli

<2-1> cadA  And pdxY  Confirmation of production effect of cadaverine according to expression

pyridoxal (PL), pyridoxal (PL), or pyridoxine (PL) to confirm the effect of producing cadaverine from lysine using Escherichia coli expressing cadA and pdxY simultaneously pyridoxine, PN) as a coenzyme, the recombinant E. coli was cultured to confirm the effect of producing cadaverine.

Concretely, the cadA and pdxY co-expressed recombinant E. coli prepared in Example <1-1> were cultured and washed with distilled water, and then 0.5 M sodium acetate buffer, pH 6.0). Substrate and coenzyme were added to the 0.5 M sodium acetate buffer solution to a final concentration of 1 M lysine and 0.2 mM PLP, PL or PN, and the final volume of the reaction solution was adjusted to 500 μl. After sufficiently suspending the reaction solution, the reaction was carried out in a constant temperature water bath at 37 캜 for 2 hours. Then, a reaction solution was obtained and analyzed by HPLC (YL9100 system, Youngin Instrument, Korea) to confirm the concentration of the produced catavalin in the reaction solution. The column for the HPLC analysis was Capcell Pack® (5 μm C18 UG 120, 4.6 × 250 mm; Shiseido Co., Ltd., Japan) and the temperature of the column was set at 35 ° C. The mobile phase solvent for the HPLC analysis was acetonitrile and 25 mM sodium acetate buffer solution (pH 4.8) were mixed according to the time gradient as shown in Table 1, and the flow rate was 1 ml / min.

Escherichia coli transformed with cadA expression plasmid alone (no expression of pdxY) was used as a negative control, and Escherichia coli transformed with a cadA expression plasmid and a pdxK expression plasmid was used as a comparative control. The reaction was carried out.

Concentration gradient conditions for HPLC mobile phase solvent for quantitative analysis of produced catarrhine Time (minutes) Acetonitrile (%) Sodium acetate buffer solution (%) 0 20 80 2 25 75 32 60 40 35 20 80

As a result, as shown in FIG. 4, when negative control using E. coli expressing cadA alone and PLD as a coenzyme in the experimental group expressing cadA and pdxY produced cadaverine effectively from lysine, cadA and pdxK were expressed In the control group using Escherichia coli was not effective in producing cadaverine. In addition, in the experimental group of cadA and pdxY-expressing E. coli, significant cadarin production was possible even when PL or PN was used as a coenzyme in addition to PLP, and cadA and pdxK using PLP as the coenzyme were comparable to the control group, (Fig. 4). In the case of the negative control, when PL or PN was added as coenzyme, it was not converted to PLP and cadA did not show activity, and cadarin production was not observed.

<2-2> on cadA  Confirmation of production effect of cadaverine according to kinds of coenzyme in Korean

it was confirmed that Escherichia coli expressing both cadA and pdxY can convert lysine to cadaverine in an environment in which PNP or PL was added as a coenzyme as well as PLP. Therefore, Escherichia coli expressing cadA and pdxY at the same time as Escherichia coli expressing cadA alone The effect of the addition of PLP precursor on the production of cadaverine was confirmed.

Concretely, the cadA and pdxY co-expressed recombinant E. coli prepared in Example <1-1> were cultured and washed with distilled water, and then 0.5 M sodium acetate buffer, pH 6.0). Substrate and coenzyme were added to the 0.5 M sodium acetate buffer solution to a final concentration of 0.4 M lysine and 0.2 mM PLP, PL or PN, and the final volume of the reaction solution was adjusted to 500 μl. After sufficiently suspending the reaction solution, the reaction was carried out in a constant temperature water bath at 37 캜 for 100 hours. From the start of the reaction, a reaction solution was obtained according to time, and HPLC analysis was carried out in the same manner as in Example <2-1> above to confirm the concentration of the produced catarrhine.

As a result, as shown in FIG. 5, it was confirmed that cadabelin was produced at a high rate in both Escherichia coli expressing cadA and pdxY and Escherichia coli expressing only cadA (FIG. 5A) in the case of PLP as a coenzyme. In addition, Escherichia coli expressing cadA alone can not produce catarrhine significantly in the absence of coenzyme, whereas in E. coli expressing pdxY and cadA, it can produce cadaverine at a significant level even though no coenzyme is added (Fig. 5B). Similarly, Escherichia coli expressing only cadA in the reaction environment in which PL or PN was added as a coenzyme showed no effect of producing cadaverine, but in E. coli expressing pdxY and cadA, PL or PN was converted to PLP by pdxY, (Fig. 5C and Fig. 5D). The results are shown in Figs. 5C and 5D.

The recombinant Escherichia coli expressing the lysine decarboxylase (cadA) and the pyridoxal kinase (pdxY) simultaneously produced from Escherichia coli according to the present invention can be effectively used for PLP recycling and production of cadaverine . Specifically, when cadA converts lysine to cadaverine, when PLP is converted to PL, pdxY can convert PL to PLP and be used again in cadA, and mass production of cadaverine capable of reusing PLP &Lt; / RTI &gt;

The PLP reuse system according to the present invention can effectively reuse the PLP even if a relatively low cost PL or PN is used as the coenzyme without using the expensive PLP required in the cataract production process through the conventional biological method The lysine can be converted into cadaverine, so that the mass production method of the cadaverine of the present invention is economical and efficient.

<110> Konkuk University Industrial Cooperation Corp          Sejong Industry-Academia Cooperation Foundation Hongik University <120> Producing method of cadaverin by recycling of          pyridoxal-5-phosphate with pyridoxal kinase <130> 1060710 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 2148 <212> DNA <213> Escherichia coli <400> 1 atgaacgtta ttgcaatatt gaatcacatg ggggtttatt ttaaagaaga acccatccgt 60 gaacttcatc gcgcgcttga acgtctgaac ttccagattg tttacccgaa cgaccgtgac 120 gacttattaa aactgatcga aaacaatgcg cgtctgtgcg gcgttatttt tgactgggat 180 aaatataatc tcgagctgtg cgaagaaatt agcaaaatga acgagaacct gccgttgtac 240 gcgttcgcta atacgtattc cactctcgat gtaagcctga atgacctgcg tttacagatt 300 agcttctttg aatatgcgct gggtgctgct gaagatattg ctaataagat caagcagacc 360 actgacgaat atatcaacac tattctgcct ccgctgacta aagcactgtt taaatatgtt 420 cgtgaaggta aatatacttt ctgtactcct ggtcacatgg gcggtactgc attccagaaa 480 gt; tccatttcag tatctgaact gggttctctg ctggatcaca gtggtccaca caaagaagca 600 gaacagtata tcgctcgcgt ctttaacgca gaccgcagct acatggtgac caacggtact 660 tccactgcga acaaaattgt tggtatgtac tctgctccag caggcagcac cattctgatt 720 gccgtaact gccacaaatc gctgacccac ctgatgatga tgagcgatgt tacgccaatc 780 tatttccgcc cgacccgtaa cgcttacggt attcttggtg gtatcccaca gagtgaattc 840 cagcacgcta ccattgctaa gcgcgtgaaa gaaacaccaa acgcaacctg gccggtacat 900 gctgtaatta ccaactctac ctatgatggt ctgctgtaca acaccgactt catcaagaaa 960 acactggatg tgaaatccat ccactttgac tccgcgtggg tgccttacac caacttctca 1020 ccgatttacg aaggtaaatg cggtatgagc ggtggccgtg tagaagggaa agtgatttac 1080 gaaacccagt ccactcacaa actgctggcg gcgttctctc aggcttccat gatccacgtt 1140 aaaggtgacg taaacgaaga aacctttaac gaagcctaca tgatgcacac caccacttct 1200 ccgcactacg gtatcgtggc gtccactgaa accgctgcgg cgatgatgaa aggcaatgca 1260 ggtaagcgtc tgatcaacgg ttctattgaa cgtgcgatca aattccgtaa agagatcaaa 1320 cgtctgagaa cggaatctga tggctggttc tttgatgtat ggcagccgga tcatatcgat 1380 acgactgaat gctggccgct gcgttctgac agcacctggc acggcttcaa aaacatcgat 1440 aacgagcaca tgtatcttga cccgatcaaa gtcaccctgc tgactccggg gatggaaaaa 1500 gcggcacca tgagcgactt tggtattccg gccagcatcg tggcgaaata cctcgacgaa 1560 catggcatcg ttgttgagaa aaccggtccg tataacctgc tgttcctgtt cagcatcggt 1620 atcgataaga ccaaagcact gagcctgctg cgtgctctga ctgactttaa acgtgcgttc 1680 gacctgaacc tgcgtgtgaa aaacatgctg ccgtctctgt atcgtgaaga tcctgaattc 1740 tatgaaaaca tgcgtattca ggaactggct cagaatatcc acaaactgat tgttcaccac 1800 aatctgccgg atctgatgta tcgcgcattt gaagtgctgc cgacgatggt aatgactccg 1860 tatgctgcat tccagaaaga gctgcacggt atgaccgaag aagtttacct cgacgaaatg 1920 gtaggtcgta ttaacgccaa tatgatcctt ccgtacccgc cgggagttcc tctggtaatg 1980 ccgggtgaaa tgatcaccga agaaagccgt ccggttctgg agttcctgca gatgctgtgt 2040 gaaatcggcg ctcactatcc gggctttgaa accgatattc acggtgcata ccgtcaggct 2100 gatggccgct ataccgttaa ggtattgaaa gaagaaagca aaaaataa 2148 <210> 2 <211> 715 <212> PRT <213> Escherichia coli <400> 2 Met Asn Val Ile Ale Ile Leu Asn His Met Gly Val Tyr Phe Lys Glu   1 5 10 15 Glu Pro Ile Arg Glu Leu His Arg Ala Leu Glu Arg Leu Asn Phe Gln              20 25 30 Ile Val Tyr Pro Asn Asp Arg Asp Asp Leu Leu Lys Leu Ile Glu Asn          35 40 45 Asn Ala Arg Leu Cys Gly Val Ile Phe Asp Trp Asp Lys Tyr Asn Leu      50 55 60 Glu Leu Cys Glu Glu Ile Ser Lys Met Asn Glu Asn Leu Pro Leu Tyr  65 70 75 80 Ala Phe Ala Asn Thr Tyr Ser Thr Leu Asp Val Ser Leu Asn Asp Leu                  85 90 95 Arg Leu Gln Ile Ser Phe Phe Glu Tyr Ala Leu Gly Ala Ala Glu Asp             100 105 110 Ile Ala Asn Lys Ile Lys Gln Thr Thr Asp Glu Tyr Ile Asn Thr Ile         115 120 125 Leu Pro Pro Leu Thr Lys Ala Leu Phe Lys Tyr Val Arg Glu Gly Lys     130 135 140 Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Phe Gln Lys 145 150 155 160 Ser Pro Val Gly Ser Leu Phe Tyr Asp Phe Phe Gly Pro Asn Thr Met                 165 170 175 Lys Ser Asp Ile Ser Ile Ser Val Ser Glu Leu Gly Ser Leu Leu Asp             180 185 190 His Ser Gly Pro His Lys Glu Ala Glu Gln Tyr Ile Ala Arg Val Phe         195 200 205 Asn Ala Asp Arg Ser Tyr Met Val Thr Asn Gly Thr Ser Thr Ala Asn     210 215 220 Lys Ile Val Gly Met Tyr Ser Ala Pro Ala Gly Ser Thr Ile Leu Ile 225 230 235 240 Asp Arg Asn Cys His Lys Ser Leu Thr His Leu Met Met Met Ser Asp                 245 250 255 Val Thr Pro Ile Tyr Phe Arg Pro Thr Arg Asn Ala Tyr Gly Ile Leu             260 265 270 Gly Ile Pro Gln Ser Glu Phe Gln His Ala Thr Ile Ala Lys Arg         275 280 285 Val Lys Glu Thr Pro Asn Ala Thr Trp Pro Val Ala Val Ile Thr     290 295 300 Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Phe Ile Lys Lys 305 310 315 320 Thr Leu Asp Val Lys Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr                 325 330 335 Thr Asn Phe Ser Pro Ile Tyr Glu Gly Lys Cys Gly Met Ser Gly Gly             340 345 350 Arg Val Glu Gly Lys Val Ile Tyr Glu Thr Gln Ser Thr His Lys Leu         355 360 365 Leu Ala Ala Phe Ser Gln Ala Ser Met Ile His Val Lys Gly Asp Val     370 375 380 Asn Glu Glu Thr Phe Asn Glu Ala Tyr Met Met His Thr Thr Thr Ser 385 390 395 400 Pro His Tyr Gly Ile Val Ala Ser Thr Glu Thr Ala Ala Ala Met Met                 405 410 415 Lys Gly Asn Ala Gly Lys Arg Leu Ile Asn Gly Ser Ile Glu Arg Ala             420 425 430 Ile Lys Phe Arg Lys Glu Ile Lys Arg Leu Arg Thr Glu Ser Asp Gly         435 440 445 Trp Phe Phe Asp Val Trp Gln Pro Asp His Ile Asp Thr Thr Glu Cys     450 455 460 Trp Pro Leu Arg Ser Asp Ser Thr Trp His Gly Phe Lys Asn Ile Asp 465 470 475 480 Asn Glu His Met Tyr Leu Asp Pro Ile Lys Val Thr Leu Leu Thr Pro                 485 490 495 Gly Met Glu Lys Asp Gly Thr Met Ser Asp Phe Gly Ile Pro Ala Ser             500 505 510 Ile Val Ala Lys Tyr Leu Asp Glu His Gly Ile Val Val Glu Lys Thr         515 520 525 Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr     530 535 540 Lys Ala Leu Ser Leu Leu Arg Ala Leu Thr Asp Phe Lys Arg Ala Phe 545 550 555 560 Asp Leu Asn Leu Arg Val Lys Asn Met Leu Pro Ser Leu Tyr Arg Glu                 565 570 575 Asp Pro Glu Phe Tyr Glu Asn Met Arg Ile Gln Glu Leu Ala Gln Asn             580 585 590 Ile His Lys Leu Ile Val His His Asn Leu Pro Asp Leu Met Tyr Arg         595 600 605 Ala Phe Glu Val Leu Pro Thr Met Val Met Thr Pro Tyr Ala Ala Phe     610 615 620 Gln Lys Glu Leu His Gly Met Thr Glu Glu Val Tyr Leu Asp Glu Met 625 630 635 640 Val Gly Arg Ile Asn Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val                 645 650 655 Pro Leu Val Met Pro Gly Glu Met Ile Thr Glu Glu Ser Arg Pro Val             660 665 670 Leu Glu Phe Leu Gln Met Leu Cys Glu Ile Gly Ala His Tyr Pro Gly         675 680 685 Phe Glu Thr Asp Ile His Gly Ala Tyr Arg Gln Ala Asp Gly Arg Tyr     690 695 700 Thr Val Lys Val Leu Lys Glu Glu Ser Lys Lys 705 710 715 <210> 3 <211> 864 <212> DNA <213> Escherichia coli <400> 3 atgatgaaaa atattctcgc tatccagtct cacgttgttt atggtcatgc gggtaacagt 60 gcggcagagt ttccgatgcg ccgcctgggc gcgaacgtct ggccgctgaa caccgttcaa 120 ttttctaatc acacccaata cggcaaatgg actggctgcg tgatgccgcc cagccattta 180 accgaaattg tgcaaggcat tgccgccatt gataaattac acacctgtga tgccgtatta 240 agtggctatc tgggatcggc ggagcagggt gaacatatcc tcggtatcgt ccgtcaggtg 300 aaagccgcga atccgcaggc gaaattatttt tgcgatccgg taatgggtca tccggaaaaa 360 ggctgtatcg ttgcaccggg tgtcgcagag tttcatgtgc ggcacggttt gcctgccagc 420 gatatcattg cgccaaatct ggttgagctg gaaatactct gtgagcatgc ggtaaataac 480 gtcgaagaag cggttctggc agcgcgcgaa ctcattgcgc aagggccaca aattgtgttg 540 gttaaacacc tggcgcgagc tggctacagc cgtgaccgtt ttgaaatgct gctggtcacc 600 gccgatgaag cctggcatat cagccgtccg ctggtggatt ttggtatgcg ccagccggta 660 ggtgttggtg atgtgacgag cggtttactg ctggtgaaac tgcttcaggg ggcaacgctg 720 caggaggcgc tggaacatgt gaccgctgca gtctacgaaa tcatggtgac caccaaagca 780 atgagagat atgagctgca agtggtggct gctcaggatc gtattgccaa accagaacat 840 tacttcagcg caacaaagct ctga 864 <210> 4 <211> 287 <212> PRT <213> Escherichia coli <400> 4 Met Met Lys Asn Ile Leu Ala Ile Gln Ser His Val Val Tyr Gly His   1 5 10 15 Ala Gly Asn Ser Ala Ala Glu Phe Pro Met Arg Arg Leu Gly Ala Asn              20 25 30 Val Trp Pro Leu Asn Thr Val Gln Phe Ser Asn His Thr Gln Tyr Gly          35 40 45 Lys Trp Thr Gly Cys Val Met Pro Ser Ser His Leu Thr Glu Ile Val      50 55 60 Gln Gly Ile Ala Ala Ile Asp Lys Leu His Thr Cys Asp Ala Val Leu  65 70 75 80 Ser Gly Tyr Leu Gly Ser Ala Glu Gln Gly Glu His Ile Leu Gly Ile                  85 90 95 Val Arg Gln Val Lys Ala Ala Asn Pro Gln Ala Lys Tyr Phe Cys Asp             100 105 110 Pro Val Met Gly His Pro Glu Lys Gly Cys Ile Val Ala Pro Gly Val         115 120 125 Ala Glu Phe His Val Arg His Gly Leu Pro Ala Ser Asp Ile Ile Ala     130 135 140 Pro Asn Leu Val Glu Leu Glu Ile Leu Cys Glu His Ala Val Asn Asn 145 150 155 160 Val Glu Glu Ala Val Leu Ala Ala Arg Glu Leu Ile Ala Gln Gly Pro                 165 170 175 Gln Ile Val Leu Val Lys His Leu Ala Arg Ala Gly Tyr Ser Arg Asp             180 185 190 Arg Phe Glu Met Leu Leu Val Thr Ala Asp Glu Ala Trp His Ile Ser         195 200 205 Arg Pro Leu Val Asp Phe Gly Met Arg Gln Pro Val Gly Val Gly Asp     210 215 220 Val Thr Ser Gly Leu Leu Leu Val Lys Leu Leu Gln Gly Ala Thr Leu 225 230 235 240 Gln Glu Val Leu Glu His Val Thr Ala Ala Val Tyr Glu Ile Met Val                 245 250 255 Thr Thr Lys Ala Met Gln Glu Tyr Glu Leu Gln Val Val Ala Ala Gln             260 265 270 Asp Arg Ile Ala Lys Pro Glu His Tyr Phe Ser Ala Thr Lys Leu         275 280 285

Claims (10)

(i) preparing a recombinant microorganism which simultaneously expresses a lysine decarboxylase and a pyridoxal kinase (PdxY) consisting of the amino acid sequence of SEQ ID NO: 4;
ii) culturing and obtaining the recombinant microorganism produced in step i);
iii) The recombinant microorganism obtained in step ii) is suspended in a reaction solution containing lysine dicarboxylase coenzyme, which is either or both of lysine substrate and pyridoxal (PL) and pyridoxine (PN) &Lt; / RTI &gt; And
iv) obtaining the cadaverine from the reaction solution reacted in step iii) above.
The method of mass production of cadaverine according to claim 1, wherein the lysine decarboxylase of step i) is cadA derived from E. coli consisting of the amino acid sequence of SEQ ID NO: 2.
delete The method of claim 1, wherein the microorganism of step i) is Escherichia coli (E. coli), Corynebacterium (Corynebacterium) or hafnia (Hafnia any one of the methods of mass, cadaverine, characterized in production medium).
delete The method of mass production of cadaverine according to claim 1, wherein the reaction of step iii) is carried out at 30 to 40 占 폚.
(i) producing a recombinant microorganism that simultaneously expresses lysine decarboxylase and a pyridoxal kinase (PdxY) consisting of the amino acid sequence of SEQ ID NO: 4;
ii) inoculating and culturing the recombinant microorganism prepared in step i) in a medium comprising lysine dicarboxylase enzyme coenzyme, which is either or both of lysine substrate and pyridoxal (PL) and pyridoxine (PN);
iii) obtaining the cadaverine from the medium cultured in the step ii).
8. The method of mass production of cadaverine according to claim 7, wherein the lysine decarboxylase of step i) is cadA derived from E. coli consisting of the amino acid sequence of SEQ ID NO: 2.
delete delete

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