KR20160125170A - GRAS strain-derived enzyme obtained by molecular evlution and GRAS Corynebaterium glutamicum having the same - Google Patents
GRAS strain-derived enzyme obtained by molecular evlution and GRAS Corynebaterium glutamicum having the same Download PDFInfo
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Abstract
Description
The present invention is derived from research carried out as part of the Global Frontier Project of the Korea Research Foundation.
[Assignment number: NRF-2012M3A6A8054887, Title: Development of biosystems resistant to nematocide using GRAS-based cellular energy]
The present invention relates to an ADP mediated activity of a phosphoenolpyruvate carboxykinase (PEPck or PCK) derived from Corynebacterium glutamicum, which is a safety-assured GRAS (Generally Recognized As Safe) strain, To a method for producing a target product with safety and a method for increasing the growth rate of cells using the same, will be.
Phosphoenolpyruvate carboxykinase (PCK) is an enzyme that mediates a reversible reaction that converts oxalacetate to phosphoenolpyruvate. Adenosine-5 'is a coenzyme, triphosphate, ATP) or guanosine-5 'triphosphate (GTP). Figure 1 shows the reversible reaction mediated by PCK. Since the reaction catalyzed by PCK is reversible, in the presence of CO 2 , PCK catalyzes the reaction to produce OAA from PEP, producing one molecule of ATP or GTP. Thus, unlike phosphoenolpyruvate carboxylase (PPC), which catalyzes the same reaction in the glycolysis pathway, an increase in the reaction by PCK may increase intracellular ATP or GTP production and concentration have. PCK is modified to use ADP as a coenzyme rather than GDP, and the expression of the gene encoding PCK is increased in the cell, or the regulatory sequence is changed so that the gene encoding PCK can be expressed in the pathway, Or by increasing the activity of PCK.
The enzyme involved in the glycolysis pathway is an enzyme that is expressed to promote glucose neogenesis when glucose is essentially lacking. Previously, the inventors have found that artificially overexpressing phosphoenolpyruvate carboxykinase in E. coli and providing a high concentration of glucose acts as a reverse-reaction activity, resulting in a 50% increase in intracellular ATP (Yeong-Deok Kwon, Sang Yup Lee, Pil Kim, 2008. 72 (4): 1138-41). In addition, we found that the productivity of succinic acid and exogenous proteins increased in high energy cells with high intracellular ATP concentrations, which were constructed in this way. (Pil Kim, Maris Laivenieks, and Claire Vieille, J Gregory Zeikus, 2004. Applied and Environmental Microbiology. 70 (2): 1238-41; Hye-Jung Kim, Yeong Deok Kwon, Sang Yup Lee, Pil Kim. 2011. Applied Microbiology and Biotechnology; 94 (4): 1079-86).
However, Escherichia coli is a non-GRAS strain in which bacterial antigens such as LPS (lipopolysaccharide) may remain in the target product, thereby requiring additional safety verification. In order to be able to utilize useful end products produced by the strains in animals including humans without additional processing, production in a GRAS strain that is considered safe is required. Accordingly, the present inventors completed the present invention by carrying out a study on a method for producing a target product and a strain capable of efficiently producing a desired product using Corynebacterium glutamicum, which is safe as a GRAS strain.
It is an object of the present invention to provide a method for the conversion of GDP-dependent phosphoenolpyruvate to oxaloacetate of wild-type phosphoenolpyruvate carboxykinase (PCK) derived from Corynebacterium glutamicum, a GRAS strain, To provide phosphoenolpyruvate carboxykinase.
It is another object of the present invention to provide a safe GRAS Corynebacterium glutamicum having a high intracellular ATP concentration.
Another object of the present invention is to provide a method for producing a target substance with safety by using GRAS Corynebacterium glutamicum having a high intracellular ATP concentration.
Another object of the present invention is to provide a method for the conversion of GDP-dependent phosphoenolpyruvate to oxaloacetate of wild-type phosphoenolpyruvate carboxykinase (PCK) derived from Corynebacterium glutamicum into ADP- The present invention provides a method for increasing the growth rate of cells by introducing a gene encoding fornonyl pyruvate carboxykinase.
In order to achieve the above-mentioned object, one aspect of the present invention relates to a pharmaceutical composition comprising, as compared with the wild type, a compound having an ADP (adenosine diphosphate) -dependent conversion activity of phosphoenolpyruvate to oxaloacetate, a Corynebacterium glutamicum- To provide phosphoenolpyruvate carboxykinase (PCK).
The phosphoenolpyruvate carboxykinase is an enzyme that mediates the conversion of phosphoenolpyruvate (PEP) produced from glucose to oxaloacetate in glycolysis, as described in FIG. 2, Use GDP or ADP as the coenzyme. In order to increase the yield and concentration of ATP production in cells for the production of the target substance, phosphoenolpyruvate carboxykinase requires ADP dependency using ADP as a coenzyme. The phosphoenolpyruvate carboxykinase derived from Corynebacterium glutamicum, which is a GRAS strain, is an enzyme which predominantly uses GDP as a coenzyme in the reaction of phosphoenolpyruvic acid to oxalacetic acid and has an activity against GDP 5 times higher than ADP. Therefore, in order to increase the intracellular ATP production yield or concentration, the GDP dependency of phosphoenolpyruvate carboxycinase derived from Corynebacterium glutamicum is subjected to molecular evolution to improve the ADP dependence, and ADP That is, phosphoenolpyruvate carboxykinase mainly using ADP as a coenzyme.
As used herein, the term " Generally Recognized As Safe " (GRAS) means a substance that is judged safe by experienced and trained professionals when they evaluate the safety under the intended use conditions of the substance through scientific procedures, Can be used as synonyms. The GRAS system is unique to the United States and applies to food and food chemicals with high safety (under certain conditions of use), but its perceptions are being recognized not only in the United States but also internationally and gradually spreading.
The terms "GDP dependent" and "ADP dependent ", as used herein in connection with phosphoenolpyruvate carboxykinase, are intended to include those in which phosphoenolpyruvate carboxykinase converts phosphoenolpyruvate carboxylic acid to oxalacetic acid Means using GDP and ADP as coenzymes in the reaction, respectively, and can be used synonymously with "GDP mediated" and "ADP mediated".
As used herein, the term "molecular evolution" refers to an evolution based on the characteristics of molecular structures, such as the base sequence of DNA having the genetic information or the amino acid sequence of the protein, and the process of artificially inducing a change in gene or protein, Lt; / RTI >
The term "high energy" as used herein in reference to a cell or strain means that the intracellular ATP concentration is high.
The phosphoenolpyruvate carboxykinase of Corynebacterium glutamicum is a GDP-dependent enzyme that predominantly uses GDP as a coenzyme, but it has been shown to undergo error-prone PCR to improve it to have ADP- (Random mutagenesis), and mutagenesis was carried out by molecular evolution, which selects the phosphoenolpyruvate carboxykinase, which has significantly elevated the ADP-mediated enzyme specific activity among these mutants PCK, 52-C10, 13-C4, and 52-G5.
In one embodiment of the present invention, the phosphoenolpyruvate carboxykinase is such that T (thymine) at position 136 of SEQ ID NO: 1 is replaced with C (cytosine) and G (guanine) at position 588 is replaced with A adenine), and a phosphoenolpyruvate carboxykinase having a sequence in which Trp (tryptophan) at position 46 of SEQ ID NO: 2 is substituted with Arg (arginine).
In another embodiment of the present invention, the phosphoenolpyruvate carboxykinase is encoded by a sequence in which G at position 733 of SEQ ID NO: 1 is substituted with A, Gly (glycine) at position 245 of SEQ ID NO: 2 Arg (arginine). ≪ / RTI >
In one embodiment of the invention, the phosphoenolpyruvate carboxykinase may be a phosphoenolpyruvate carboxykinase encoded by a sequence lacking C at position 342 of SEQ ID NO: 1.
In one embodiment of the present invention, the phosphoenolpyruvate carboxykinase with increased ADP dependency compared to the wild type is shown in Figure 1 in which the conversion of phosphoenol pyruvate carboxylic acid to oxalacetic acid is reduced by ADP to ATP , Thereby contributing to an increase in intracellular ATP production yield or concentration.
In one embodiment of the invention, the wild type phosphoenolpyruvate carboxykinase may be a phosphoenolpyruvate carboxykinase encoded by SEQ ID NO: 1 of Corynebacterium glutamicum ATCC 13032.
One aspect of the invention relates to a method of increasing the rate of growth of a cell, comprising transforming the cell to have an increased intracellular ATP concentration relative to the parent cell.
In one embodiment of the invention, the cell may be one wherein the expression of a gene encoding phosphoenolpyruvate carboxykinase (PCK) versus a parent cell is modified to increase intracellular ATP concentration.
In one embodiment of the present invention, in one embodiment of the invention, the expression of the gene encoding the phosphoenolpyruvate carboxykinase (PCK) in a cell having increased intracellular ATP concentration relative to the parental cell is in a cell Or may be modified to increase the intracellular ATP concentration by being regulated to be expressed in the pathway.
In one embodiment of the present invention, the cell has an intracellular ATP concentration by increasing the number of copies of the gene encoding phosphoenolpyruvate carboxykinase, or by modifying the expression control sequence or coding sequence of the gene . ≪ / RTI >
In one embodiment of the present invention, the cell is a wild-type, phosphoenolpyruvate-derived phosphoenolate derived from Corynebacterium glutamicum, which has increased ADP (adenosine diphosphate) -dependent conversion activity of oxaloacetate to phosphoenolpyruvate It may be modified by introducing a gene encoding a pyruvate carboxykinase into the cell.
The recombinant cell into which the gene coding for phosphoenolpyruvate carboxykinase derived from Corynebacterium glutamicum, which has increased ADP-dependent activity of phosphoenolpyruvate to oxaloacetate, Compared with parent cells, they have a higher intracellular ATP concentration and an increased growth rate.
The phosphoenolpyruvate carboxykinase is prepared by substituting C (cytosine) for T (thymine) at position 136 of SEQ ID NO: 1 and substituting G (guanine) at position 588 for A And can be a phosphoenolpyruvate carboxykinase having a sequence in which Trp (tryptophan) at position 46 of SEQ ID NO: 2 is substituted with Arg (arginine).
In another embodiment of the present invention, the phosphoenolpyruvate carboxykinase is encoded by a sequence in which G at position 733 of SEQ ID NO: 1 is substituted with A, Gly (glycine) at position 245 of SEQ ID NO: 2 Arg (arginine). ≪ / RTI >
In one embodiment of the invention, the phosphoenolpyruvate carboxykinase may be a phosphoenolpyruvate carboxykinase encoded by a sequence lacking C at position 342 of SEQ ID NO: 1.
In one embodiment of the invention, the cells may be bacteria, yeast, fungi, plant cells or animal cells.
In one embodiment of the invention, the cell may be Corynebacterium glutamicum.
In one embodiment of the present invention, the introduction of the gene encoding the phosphoenolpyruvate carboxykinase includes, but is not limited to, transformation or transfection, and may be performed using conventional methods known to those skilled in the art ≪ / RTI >
One aspect of the present invention is to provide a method of inhibiting ADP-dependent conversion of phosphoenolpyruvate to oxaloacetate, which has an increased intracellular ATP concentration compared to the parent strain, which has phosphoenolpyruvate carboxykinase, Lt; RTI ID = 0.0 > Corynebacterium glutamicum. ≪ / RTI >
As used herein, the term "parent strain" means that a gene coding for phosphoenolpyruvate carboxykinase (PCK) is modified by a molecular biology technique such as a mutation or recombinant method, For example, a strain in which the ADP dependency is enhanced and the intracellular ATP production yield or concentration is increased.
In one embodiment of the invention, the recombinant Corynebacterium glutamicum is encoded by a phosphoenolpyruvate carboxykinase, which has increased ADP-dependent conversion of phosphoenolpyruvate to oxaloacetate as compared to the wild type, May be a strain introduced by a method of introducing a foreign gene, including, but not limited to, transformation or transduction.
In one embodiment of the invention, the recombinant Corynebacterium glutamicum is an is the number 136 position of SEQ ID NO: 1 T substituted by a C and a G of times 588 position of SEQ ID NO: 1 is substituted by A sequence (cgl2863 - 52- C10) may be recombinant in Corynebacterium kumil having a pyruvate carboxy kinase play a phospholipase encoded by the.
In one embodiment of the invention, the recombinant Corynebacterium glutamicum is a G in the 733 position of SEQ ID No. 1 encoded by a sequence (cgl2863 -13- C4) substituted with A phosphorylation play pyruvate Or a recombinant Corynebacterium glutamicum having a carboxykinase.
In one embodiment of the invention, the recombinant Corynebacterium glutamicum is a by the time of the 342 position of SEQ ID NO: 1 C deletion sequence (cgl2863 -52- G5) coding phosphorylation play pyruvate carboxy kinase Lt; RTI ID = 0.0 > Corynebacterium glutamicum. ≪ / RTI >
In one embodiment of the present invention, the recombinant Corynebacterium glutamicum is produced by an increased expression of cgl2863-52 -C10 , which encodes an improved phosphoenolpyruvate carboxykinase as compared to the parent strain, The increase in the intracellular ATP amount can be shown by the increase of the enzyme activity produced by the enzyme.
In one embodiment of the present invention, the recombinant Corynebacterium glutamicum is produced by increasing the expression of cgl2863 -13-C4 , which encodes an improved phosphoenolpyruvate carboxykinase, as compared to the parent strain, The increase in the intracellular ATP amount can be shown by the increase of the enzyme activity produced by the enzyme.
In one embodiment of the present invention, the recombinant Corynebacterium glutamicum is produced by an increased expression of cgl2863-52 -G5 , which encodes an improved phosphoenolpyruvate carboxykinase as compared to the parent strain, The increase in the intracellular ATP amount can be shown by the increase of the enzyme activity produced by the enzyme.
In one embodiment of the present invention, the parent strain may be Corynebacterium glutamicum ATCC 13032.
One aspect of the present invention relates to the use of a compound of formula I, wherein the increased intracellular ATP concentration relative to the parental strain, with phosphoenolpyruvate carboxykinase, which has increased ADP-dependent conversion activity of phosphoenolpyruvate to oxaloacetate And culturing the recombinant Corynebacterium glutamicum having the amino acid sequence of the recombinant Corynebacterium glutamicum in a condition suitable for producing the target substance.
In one embodiment of the present invention, the method further comprises the step of introducing a gene coding for the target substance into the recombinant Corynebacterium glutamicum from the outside or culturing the recombinant Corynebacterium glutamicum in a metabolic pathway Lt; RTI ID = 0.0 > genetically < / RTI >
In one embodiment of the present invention, the target substance may be a protein, an amino acid, a nucleic acid, an antibiotic, or a metabolite.
In one embodiment of the invention, the recombinant Corynebacterium glutamicum may be derived from Corynebacterium glutamicum ATCC 13032.
As used herein, the term "target substance" means a substance produced by a method comprising culturing a cell according to one embodiment of the present invention, and includes a protein, an L-amino acid, a nucleic acid, an antibiotic, a vitamin, , Metabolites, but are not limited thereto.
In the present specification, "genetically transforming to produce a target substance" means that a gene necessary for biosynthesis of a target substance is externally introduced into a microorganism, or expression of a gene involved in the production of a target substance existing in the genome of the cell Or inhibiting or eliminating the expression of a gene involved in the degradation of a target substance or transforming the cell into a state favorable for producing a target substance.
The method for producing a target substance according to the present invention comprises culturing a recombinant Corynebacterium sp. Microorganism under conditions suitable for producing a target substance.
The step of culturing the cells may be carried out under culture conditions and with a suitable medium known in the art. Conditions suitable for producing the target substance can be easily determined by the person skilled in the art depending on the target substance and the cell. For example, when induction is required for expression of a gene encoding a target substance introduced from the outside, the step of culturing the cell may further include inducing expression of the gene. Cultivation includes, but is not limited to, batch, continuous, and fed-batch cultivation.
The method for producing a target substance according to the present invention may further comprise the step of recovering a target substance from a culture medium of a cell. The step of recovering the target substance from the culture can be carried out by a conventional method known in the art. Methods such as centrifugation, filtration, chromatography, crystallization and the like may be used to separate the target substance, but the present invention is not limited thereto.
Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are intended to illustrate the invention and should not be construed as limiting the invention.
The phosphoenolpyruvate carboxykinase derived from Corynebacterium glutamicum according to one embodiment of the present invention has an activity of mediating the ADP-dependent response of phosphoenolpyruvate to oxalacetic acid is higher than that of the wild type, Using recombinant Corynebacterium glutamicum having a high intracellular ATP level, obtained by expressing phosphoenolpyruvate carboxykinase in Corynebacterium glutamicum, which is a GRAS strain, Expression, biosynthesis, and material transport, it is possible to efficiently produce useful and safe target substances including proteins, metabolites and the like.
1 is a schematic diagram showing the reaction of phosphoenolpyruvate carboxykinase.
Figure 2 shows the reaction catalyzed by phosphoenolpyruvate carboxykinase (PCK) and phosphoenolpyruvate carboxylase (PPC) in E. coli.
FIG. 3 is a graph showing the results obtained by comparing the cells of high energy Corynebacterium glutamicum (+ 52-C10, +13-C4, and +52-G5) and control (C. glutamicum, + WT PEPck) My ATP concentration. "+" Indicates that WT PEPck, improved PCK 52-C10, 13-C4, and 52-C5, respectively, were introduced into the control Corynebacterium glutamicum.
Figure 4 is the concentration of high energy Corynebacterium glutamicum coexpressed with lysine exogenous transport protein and lysine transported outside the cells of a control, according to one embodiment of the present invention.
Figures 5a to 5e are each plasmid pSL360 (pSK1CatP180), pSL (P 180 - cgl2863 -52-C10), pSL (P 180 - cgl2863 -13- C4), pSL (P 180 - cgl2863 -52- G5), and pSL ( P < 180 & gt; - lysE ).
Figure 6 shows the growth curves of the recombinant Corynebacterium glutamicum CgPck52-C10 and the control group according to one embodiment of the present invention.
Example One. Corynebacterium Glutamicum origin Phosphoenolpyruvate Carboxykinase Improvement by molecular evolution
Corynebacterium < RTI ID = 0.0 > glutamicum ) ATCC 13032 derived phosphoenolpyruvate carboxykinase (PCK) was used to dephosphorylate phosphoenolpyruvate with oxalacetate to allow the use of ADP rather than GDP as a coenzyme to accept phosphate groups better than the wild type, Molecular evolution was performed on the gene coding for PCK ( cgl2863 , GenBank ID: BA000036.3, SEQ ID NO: 1) to improve ADP dependence.
Random mutagenesis (Error-prone PCR) was performed using the wild-type phosphoenolpyruvate carboxykinase gene cgl2863 as a template in combination with pET28a (Merck KGaA, Darmstadt, Germany) under the control of T7 promoter Random mutagenesis was performed. The forward and reverse primers used for the PCR were T7 promoter (5'-TAATACGACTCACTATAGGG-3 ': SEQ ID NO: 5) and T7 terminator promoter (5' -GCTAGTTATTGCTCAGCGG-3 ': SEQ ID NO: 6). In order to construct 10,520 mutant libraries through real-time PCR and to select phosphoenolpyruvate carboxykinase with improved ADP mediated activity, ADP mediated activities were measured and screened as follows.
The mutant protein to be analyzed is disrupted and the solution is treated with phosphoenol pyruvate carboxykinase enzyme substrate phosphoenolpyruvate, ADP, MgSO 4 and 50 mM 4- (2-hydroxyethyl) -1-piperazine (HEPES, Acros organics-Thermo Fisher Scientific, Geel, Belgium) (pH 7.0) buffer solution, and the mixture was reacted for 10 minutes. (ATP) Bioluminescent Assay Kit for ATP Quantitation (Sigma-Aldrich, St. Louis, Mo., USA) was used to screen mutants with increased amounts of ADP to ATP, CA), and the luminescence was measured in the obtained solution using a luminometer (1420 multilabel counter VICTOR3, PerkinElmer, Waltham, Mass., USA). Mutants with higher ADP mediated activity were selected by selecting mutants with higher measured luminescence intensity than wild type mutants.
In addition, malate dehydrogenase binding assay (Malate Dehydrogenase Coupling Assay) was performed for more accurate activity measurement of selected mutants. This is a method for quantifying the amount of oxalacetic acid produced by the reaction of phosphoenolpyruvate carboxykinase, wherein the malic acid dehydrogenase is an enzyme that converts oxalacetic acid to malic acid. In this process, NADH (Diphosphopyridine nucleotide, reduced form) is used as a coenzyme. At this time, a certain amount of NADH coenzyme can be added, and the concentration of oxalacetic acid can be estimated by measuring the absorbance at 340 nm of NADH reduction amount. After the enzyme substrate reaction of the phosphoenolpyruvate carboxykinase as described above, 100 μl of the substrate reaction mixture and 5 μl of Malic dehydrogenase (Malic Dehydrogenase from porcine heart, Sigma-Aldrich, St. Louis, Mo., USA, CA) And 0.14 mM of NADH (β-Nicotinamide adenine dinucleotide, reduced disodium salt hydrate, Sigma-Aldrich, St. Louis, Mo., USA) were mixed and reacted for 20 minutes. Then, the absorbance at 340 nm was measured to estimate the amount of oxalacetic acid in the substrate reaction solution. A standard curve for the reduction of NADH by oxalacetic acid concentration was prepared by using oxaloacetate (Sigma-Aldrich, St. Louis, MO, USA, CA) as a reference material.
This resulted in the selection of phosphoenolpyruvate carboxykinases 52-C10, 13-C4, and 52-G5 with improved ADP mediated activity.
The enzyme inactivity on ADP substrate of 52-C10 was 0.231 mmole-oxalacetic acid / mg-protein / min, the enzyme inactivity on 13-C4 ADP substrate was 0.221 mmole-oxalacetic acid / mg-protein / min, 52-G5 Enzyme activity on ADP substrate was measured as 0.201 mmole-oxalacetic acid / mg-protein / min. This is an increase of 51%, 44%, and 15%, respectively, compared with the wild-type enzyme inactive 0.153 mmole-oxalacetic acid / mg-protein / min. Table 1 below summarizes the results of activity measurement.
Contrast active
Contrast active
Sequences of 52-C10, 13-C4, and 52-G5 were identified through sequencing (Biomedic, Bucheon, Korea).
52-C10 sequence changes of the gene in two cases, T is the time position 136 of SEQ ID NO: 1 is replaced with C, was substituted for the A times 588 position to G (cgl2863 -52- C10), changes in the amino acid sequence , And Trp (tryptophan) of SEQ ID NO: 2 was substituted with Arg (arginine).
In the case of 13-C4, the sequence change of the gene is 1, the G at position 733 of SEQ ID NO: 1 is substituted with A ( cgl2863 -13- C4 ), the amino acid sequence change is 1, Gly (Glycine) was replaced with Arg (Arginine).
For a 52-G5 had the amino acid sequence of 342 times C (Cytosine) of SEQ ID NO: 1 is deleted has changed is opened decrypted frame (cgl2863 -52- G5), SEQ ID NO: 3.
Example 2. Improved Corynebacterium Glutamicum origin Phosphoenolpyruvate Carboxykinase Increased intracellular energy through expression Corynebacterium Glutami Kum Co.
Intracellular ATP amount is increased Corynebacterium glutamicum for the production of Example 1, the genes of the play to an improved phospholipase pyruvate carboxy kinase obtained from transformed with a plasmid having (cgl2863 -52- C10) was Recombinant Corynebacterium glutamicum CgPck52-C10 was prepared as follows. Transformed with a plasmid having a cgl2863 -13- C4 in the same manner in which switch recombinant Corynebacterium glutamicum CgPck13-C4, cgl2863 recombinant was transformed with a plasmid having -52- G5 Corynebacterium glutamicum CgPck52-G5 .
2-1. CgPck52 - C10 Production of strain
(1) Preparation of recombinant plasmids containing cgl2863 -52- C10
Obtained in Example 1, genes play an enhanced phosphorylation pyruvate carboxy kinase (cgl2863 -52- C10) received treated with Pst I, and the deposit from the Korea University professor yiheungsik, P180 promoter regulatory under pSL360 (pSK1CatP180) ( Park Soo-Dong, Sang-Nam Lee, Ik-Hyun Park, Jong-Su Choi, Wol-Kyu Jeong, Younhee Kim and Heung-Shick Lee, 2004, J. Microbiol. Biotechnol. 14 (4), 789-795) and then treated with Pst I, and these ligation pSL - were also prepared the (P 180 cgl2863 -52- C10). It shows the structure of - (cgl2863 -52- C10 P 180) Figures 5a and 5b are each pSL360 (pSK1CatP180) and pSL.
Check - (cgl2863 -52- P 180 C10) and confirmed by sequence analysis (Bio Medic, Bucheon, Republic of Korea) of the DNA fragment (1.9kb and 6.4kb) generated by treatment with Pst I constructed a recombinant plasmid pSL Respectively.
(2) transformation by pSL (P 180 cgl2863 -52- C10)
The pSL constructed in the above-mentioned (1) by using a (P 180 C10 cgl2863 -52-) the Electro Cell Manipulator -ECM630 (BTX Harvard Apparatus , Holliston, Massachusetts, USA) in the electroporation of Corynebacterium glutamicum ATCC by transforming the 13032, the recombinant of Corynebacterium glutamicum ATCC 13032 / pSL (P 180 - cgl2863 -52-C10) expressing cgl2863 -52-C10 was prepared.
2-2. CgPck13 - C4 Production of strain
(1) Preparation of recombinant plasmids containing cgl2863 -13- C4
Obtained in Example 1, and handles the gene (cgl2863- 13- C4) of the play to an improved phospholipase pyruvate carboxy kinase with Pst I, P180 promoter regulatory under pSL360 (pSK1CatP180) (Park Soo- Dong, Nam-Sang, and 14, (4), 789-795). After treatment with Pst I, the cells were treated with Pst I, These were ligated to construct pSL ( P 180 -cgl2863-13-C4 ). FIG. 5C shows the structure of pSL ( P 180 - cgl 2863 - 13 - C 4 ).
The constructed recombinant plasmid pSL ( P 180 cgl2863 -13- C4 ) was confirmed by identification and sequencing of the DNA fragments (1.9 kb and 6.4 kb) produced by treatment with Pst I (Biodec, Bucheon, Korea) .
(2) Transformation by pSL ( P 180 cgl2863 -13- C4 )
The pSL ( P 180 - cgl2863 - 13 - C4 ) prepared in the above (1) was electrophoresed using an Electro Cell Manipulator - ECM630 (BTX Harvard Apparatus, Holliston, Massachusetts, USA) to prepare Corynebacterium glutamicum ATCC by transforming the 13032, the recombinant of Corynebacterium glutamicum ATCC 13032 / pSL expressing cgl2863 -13- C4 - was prepared (P 180 cgl2863 -13- C4).
2-3. CgPck52 - G5 Production of strain
(1) cgl2863 - constructing a recombinant plasmid containing a 52-G5
(Cgl2863-52-G5) of the improved phosphoenolpyruvate carboxykinase obtained in Example 1 was treated with Pst I and pSL360 (pSK1CatP180) (Park Soo-Dong, Sang-Nam 14, (4), 789-795). After treatment with Pst I, the cells were treated with Pst I, These were ligated to construct pSL ( P 180 -cgl2863-52 -G5). Figure 5d shows the structure of pSL ( P 180 - cgl2863 - 52 - G5).
The constructed recombinant plasmids were identified by identification and sequence analysis (Biomedic, Bucheon, Korea) of DNA fragments (1.9 kb and 6.4 kb) produced by treatment with Pst I.
(2) Transformation by pSL ( P 180 - cgl2863 - 52 - G5)
The pSL ( P 180 - cgl2863 - 52 - G5) prepared in the above (1) was electrophoresed using an electro cell manipulator ECM630 (BTX Harvard Apparatus, Holliston, Massachusetts, USA) to prepare Corynebacterium glutamicum ATCC by transforming the 13032, cgl2863 - to prepare a recombinant Corynebacterium glutamicum ATCC 13032 / pSL expressing 52-G5 (52-G5 P 180 - - cgl2863).
2-4. Nose with increased intracellular energy Rene Bacterium Glutamic Culture and intracellular ATP Compare volumes
The absorbance (OD) at 600 nm was measured to determine biomass, and 1 OD. = 0.36 g / l was used to calculate the dry cell weight (dcw).
ATP concentration was determined using the Adenosine 5'-triphosphate (ATP) Bioluminescent Assay Kit for ATP Quantitation, Sigma-Aldrich, St. Louis, MO. Louis, MO, USA, CA) according to the manufacturer's instructions.
(1) Culture of Recombinant Corynebacterium glutamicum
For the cultivation of the recombinant Corynebacterium glutamicum prepared by 2-1, 2-2, and 2-3, 37 g of BHI (Brain-Heart Infusion) medium per liter and 9 g of glucose per liter were added Medium was used. 30 [mu] g / ml of kanamycin, a selective antibiotic of the vector contained in the recombinant Corynebacterium glutamicum, was added.
A single colony of the recombinant Corynebacterium glutamicum to be cultured was inoculated into a 15-ml test tube containing 3-ml of glucose-supplemented medium in BHI and cultured in a rotary shaking incubator (30 DEG C, 220 rpm) for 16 hours . 1 ml of the obtained culture was inoculated into a 500 ml Erlenmeyer baffled-flask containing 100 ml of glucose-containing medium and cultured at 30 ° C and 220 rpm. When the optical density of the cell culture solution measured at 600 nm reached 1.5, the cell culture solution was collected.
6 and Table 2 show the growth curves of the recombinant Corynebacterium glutamicum CgPck52-C10 and the control group. The specific grwoth rate of CgPck52-C10 was 0.748 h-1, which was increased by about 33% compared to 0.563 h-1 of the control group in which the pSL360 empty vector was introduced. This shows that the intracellular ATP increase contributed to the growth rate, as compared with the non-growth rate of 0.959 h-1, which is the strain of E. coli-derived phosphoenolpyruvate carboxykinase, EcPck.
Corynebacterium glutamicum ATCC 13032
(2) Measurement of intracellular ATP level
The cells were collected from 1-ml of the collected cell culture by centrifugation (13,000 g, 4 ° C) and suspended in 1-ml of 20-mM Tris-HCl (pH 7.0). The screw cap microtube was filled with 0.2 g of acid-washed glass beads (212-300 microns), and then 800 μl of the cell suspension was added. The cell suspension was vortexed for 6 minutes using a mini-bead beater (Model 607, Biospec product, Bartlesville, USA) while cooling on ice. The crushed suspension was centrifuged several times at 13,000 g at 4 ° C until the precipitate was completely removed to remove cell lysate, and the supernatant was used for ATP concentration analysis. ATP concentration was estimated by measuring the degree of luminescence with a luminometer using a standard substance, Adenosine 5'-Triphosphate (Sigma-Aldrich, St. Louis, Mo., USA, CA).
Figure 3 shows the result. The ATP concentration in CgPck52-C10 was 1.01 μmol / g-dcw, the ATP concentration in CgPck13-C4 was 0.87 μmol / g-dcw and the ATP concentration in CgPck52-G5 was 0.81 μmol / g-dcw. 89% and 76% higher than the ATP concentration of 0.46 μmol / g-dcw of CgPck_WT in wild-type cgl2863, respectively. The intracellular ATP concentration of the control group in which the pSL360 empty vector was introduced was 0.31 μmol / g-dcw.
Example 3. Intracellular Increased energy Corynebacterium From Glutamicum Promoting the Effect of Lysine External Transport Proteins
LysE, a protein that transports lysine to the outside of the cell, was expressed together with the recombinant Corynebacterium glutamicum obtained in Example 2 to examine the cellular energy increase and its application.
(5'-GT CCT AGG TTA AGT ACT TCC ATA G-3 ': SEQ ID NO: 7) with the genomic DNA of Corynebacterium glutamicum ATCC 13032 as a template as a lysE gene (SEQ ID NO: 4) And reverse primer (5'-CCT AGG CTA ACC CAT CAA CAT CAG-3 ': SEQ ID NO: 8). The recombinant plasmids prepared in Examples 2-1, 2-2 and 2-3 were treated with Avr II and the amplified lysE was treated with Avr II and ligated to obtain pSL ( P 180 - cgl2863 -52 - it was also prepared the cgl2863 -52- G5, lysE) - C10 , lysE), pSL (P 180 - cgl2863 -13- C4, lysE), and pSL (P 180. Recombinant Corynebacterium glutamicum was transformed into Corynebacterium glutamicum ATCC 13032 through electroporation using an Electro Cell Manipulator to prepare the recombinant Corynebacterium glutamicum. The obtained recombinant Corynebacterium glutamicum was cultured to confirm the change in the amount of lysine outside the cells.
PSL as a control - the lysE amplified by pSL360 plasmids and PCR to construct (P 180 lysE) plasmid was ligated them by treatment with Pst I. 5E shows the structure of pSL ( P 180 - lysE ). The obtained recombinant plasmid was transformed into Corynebacterium glutamicum ATCC 13032 to prepare a control Corynebacterium glutamicum which overexpresses only LysE.
3-1. Recombination Corynebacterium Glutamicum culture
For the cultivation of Corynebacterium glutamicum, medium prepared by adding 37 g of BHI (Brain-Heart Infusion) medium per liter and 9 g of glucose per liter was used. 30 [mu] g / ml of kanamycin, a selective antibiotic of the vector contained in the recombinant Corynebacterium glutamicum, was added.
A single colony of the recombinant Corynebacterium glutamicum to be cultured was inoculated into a 15-ml test tube containing 3-ml of medium containing glucose (37 g / L) added to BHI, and cultured in a rotary shaking incubator (30 ° C, 220 rpm) Lt; / RTI > for 16 hours. 1 ml of the obtained culture was inoculated into 500 ml Erlenmeyer baffled-flask containing 100 ml of glucose-supplemented medium and cultured at 30 DEG C and 220 rpm for 12 hours. Samples were taken every 2 hours for analysis of biomass quantity and lysine change.
3-2. Effect of simultaneous expression
Lysine external transport proteins use ATP energy for transport because they reverse the concentration gradient in the transport process. Therefore, it was expected that Corynebacterium with a high level of intracellular ATP would enhance the effect of lysine external transport protein.
Comparison of the amount of lysine transported outside the cell
The cell culture obtained in 3-1 was centrifuged (13,000 g, 4 캜), and the resulting supernatant was separated and used for lysine analysis. Lysine was analyzed by High Performance Liquid Chromatography equipped with a Waters 474 fluorescence detector (Waters Cooperation, Milford, Mass., USA). The lysine in the obtained supernatant was analyzed after derivatization with o-phthalaldialdehyde. (Solvent A: 80% sodium phosphate buffer solution (pH 5.5) 80%, methanol 19%,
The result is shown in Fig. The concentrations of lysine transported to the outside of the cells shown in FIG. 4 were 2.27 mmol / L in Pck52-C10-lysE, 2.10 mmol / L in Pck13-C4-lysE and 2.15 mmol / L in Pck52-G5-lysE. 1.83 mmol / L for the lysE-only control group and 1.84 mmol / L for the wild-type Pck-lysE expression. Thus, Pck52-C10-lysE, Pck13-C4-lysE, and Pck52-G5-lysE increased the external transport of lysine by 24%, 14%, and 17%, respectively,
<110> Industry-Academic Cooperation Foundation of Catholic University <120> GRAS strain-derived enzyme obtained by molecular evolution and GRAS Corynebaterium glutamicum having the same <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 1833 <212> DNA <213> Corynebacterium glutamicum ATCC 13032 <220> <221> gene ≪ 222 > (1) .. (1833) <223> cgl2863 <400> 1 atgactactg ctgcaatcag gggccttcag ggcgaggcgc cgaccaagaa taaggaactg 60 ctgaactgga tcgcagacgc cgtcgagctc ttccagcctg aggctgttgt gttcgttgat 120 ggatcccagg ctgagtggga tcgcatggcg gaggatcttg ttgaagccgg taccctcatc 180 aagctcaacg aggaaaagcg tccgaacagc tacctagctc gttccaaccc atctgacgtt 240 gcgcgcgttg agtcccgcac cttcatctgc tccgagaagg aagaagatgc tggcccaacc 300 aacaactggg ctccaccaca ggcaatgaag gacgaaatgt ccaagcatta cgctggttcc 360 atgaaggggc gcaccatgta cgtcgtgcct ttctgcatgg gtccaatcag cgatccggac 420 cctaagcttg gtgtgcagct cactgactcc gagtacgttg tcatgtccat gcgcatcatg 480 acccgcatgg gtattgaagc gctggacaag atcggcgcga acggcagctt cgtcaggtgc 540 ctccactccg ttggtgctcc tttggagcca ggccaggaag acgttgcatg gccttgcaac 600 gacaccaagt acatcaccca gttcccagag accaaggaaa tttggtccta cggttccggc 660 tcggcggaa acgcaatcct ggcaaagaag tgctacgcac tgcgtatcgc atctgtcatg 720 gctcgcgaag aaggatggat ggctgagcac atgctcatcc tgaagctgat caacccagag 780 ggcaaggcgt accacatcgc agcagcattc ccatctgctt gtggcaagac caacctcgcc 840 atgatcactc caaccatccc aggctggacc gctcaggttg ttggcgacga catcgcttgg 900 ctgaagctgc gcgaggacgg cctctacgca gttaacccag aaaatggttt cttcggtgtt 960 gctccaggca ccaactacgc atccaaccca atcgcgatga agaccatgga accaggcaac 1020 accctgttca ccaacgtggc actcaccgac gacggcgaca tctggtggga aggcatggac 1080 ggcgacgccc cagctcacct cattgactgg atgggcaacg actggacccc agagtccgac 1140 gaaaacgctg ctcaccctaa ctcccgttac tgcgtagcaa tcgaccagtc cccagcagca 1200 gcacctgagt tcaacgactg ggaaggcgtc aagatcgacg caatcctctt cggtggacgt 1260 cgcgcagaca ccgtcccact ggttacccag acctacgact gggagcacgg caccatggtt 1320 ggtgcactgc tcgcatccgg tcagaccgca gcttccgcag aagcaaaggt cggcacactc 1380 cgccacgacc caatggcaat gctcccattc attggctaca acgctggtga atacctgcag 1440 aactggattg acatgggtaa caagggtggc gacaagatgc catccatctt cctggtcaac 1500 tggttccgcc gtggcgaaga tggacgcttc ctgtggcctg gcttcggcga caactctcgc 1560 gttctgaagt gggtcatcga ccgcatcgaa ggccacgttg gcgcagacga gaccgttgtt 1620 ggacacaccg ctaaggccga agacctcgac ctcgacggcc tcgacacccc aattgaggat 1680 gtcaaggaag cactgaccgc tcctgcagag cagtgggcaa acgacgttga agacaacgcc 1740 gagtacctca ctttcctcgg accacgtgtt cctgcagagg ttcacagcca gttcgatgct 1800 ctgaaggccc gcatttcagc agctcacgct taa 1833 <210> 2 <211> 610 <212> PRT <213> Corynebaterium glutamicum ATCC 13032 <220> <221> PEPTIDE <222> (1). (610) <223> Phosphenolopyruvate carboxykinase <400> 2 Met Thr Thr Ala Ala Ile Arg Gly Leu Gln Gly Glu Ala Pro Thr Lys 1 5 10 15 Asn Lys Glu Leu Leu Asn Trp Ile Ala Asp Ala Val Glu Leu Phe Gln 20 25 30 Pro Glu Ala Val Val Phe Val Asp Gly Ser Gln Ala Glu Trp Asp Arg 35 40 45 Met Ala Glu Asp Leu Val Glu Ala Gly Thr Leu Ile Lys Leu Asn Glu 50 55 60 Glu Lys Arg Pro Asn Ser Tyr Leu Ala Arg Ser Asn Pro Ser Serp Val 65 70 75 80 Ala Arg Val Glu Ser Arg Thr Phe Ile Cys Ser Glu Lys Glu Glu Asp 85 90 95 Ala Gly Pro Thr Asn Asn Trp Ala Pro Pro Gln Ala Met Lys Asp Glu 100 105 110 Met Ser Lys His Tyr Ala Gly Ser Met Lys Gly Arg Thr Met Tyr Val 115 120 125 Val Pro Phe Cys Met Gly Pro Ile Ser Asp Pro Asp Pro Lys Leu Gly 130 135 140 Val Gln Leu Thr Asp Ser Glu Tyr Val Val Met Ser Ser Met Arg Ile Met 145 150 155 160 Thr Arg Met Gly Ile Glu Ala Leu Asp Lys Ile Gly Ala Asn Gly Ser 165 170 175 Phe Val Arg Cys Leu His Ser Val Gly Ala Pro Leu Glu Pro Gly Gln 180 185 190 Glu Asp Val Ala Trp Pro Cys Asn Asp Thr Lys Tyr Ile Thr Gln Phe 195 200 205 Pro Glu Thr Lys Glu Ile Trp Ser Tyr Gly Ser Gly Tyr Gly Gly Asn 210 215 220 Ala Ile Leu Ala Lys Lys Cys Tyr Ala Leu Arg Ile Ala Ser Val Met 225 230 235 240 Ala Arg Glu Glu Gly Trp Met Ala Glu His Met Leu Ile Leu Lys Leu 245 250 255 Ile Asn Pro Glu Gly Lys Ala Tyr His Ile Ala Ala Ala Phe Pro Ser 260 265 270 Ala Cys Gly Lys Thr Asn Leu Ala Met Ile Thr Pro Thr Ile Pro Gly 275 280 285 Trp Thr Ala Gln Val Val Gly Asp Asp Ile Ala Trp Leu Lys Leu Arg 290 295 300 Glu Asp Gly Leu Tyr Ala Val Asn Pro Glu Asn Gly Phe Phe Gly Val 305 310 315 320 Ala Pro Gly Thr Asn Tyr Ala Ser Asn Pro Ile Ala Met Lys Thr Met 325 330 335 Glu Pro Gly Asn Thr Leu Phe Thr Asn Val Ala Leu Thr Asp Asp Gly 340 345 350 Asp Ile Trp Trp Glu Gly Met Asp Gly Asp Ala Pro Ala His Leu Ile 355 360 365 Asp Trp Met Gly Asn Asp Trp Thr Pro Glu Ser Asp Glu Asn Ala Ala 370 375 380 His Pro Asn Ser Arg Tyr Cys Val Ala Ile Asp Gln Ser Pro Ala Ala 385 390 395 400 Ala Pro Glu Phe Asn Asp Trp Glu Gly Val Lys Ile Asp Ala Ile Leu 405 410 415 Phe Gly Gly Arg Arg Ala Asp Thr Val Pro Leu Val Thr Gln Thr Tyr 420 425 430 Asp Trp Glu His Gly Thr Met Val Gly Ala Leu Leu Ala Ser Gly Gln 435 440 445 Thr Ala Ala Ser Ala Glu Ala Lys Val Gly Thr Leu Arg His Hisp Pro 450 455 460 Met Ala Met Leu Pro Phe Ile Gly Tyr Asn Ala Gly Glu Tyr Leu Gln 465 470 475 480 Asn Trp Ile Asp Met Gly Asn Lys Gly Gly Asp Lys Met Pro Ser Ile 485 490 495 Phe Leu Val Asn Trp Phe Arg Arg Gly Glu Asp Gly Arg Phe Leu Trp 500 505 510 Pro Gly Phe Gly Asp Asn Ser Arg Val Leu Lys Trp Val Ile Asp Arg 515 520 525 Ile Glu Gly His Val Gly Ala Asp Glu Thr Val Val Gly His Thr Ala 530 535 540 Lys Ala Glu Asp Leu Asp Leu Asp Gly Leu Asp Thr Pro Ile Glu Asp 545 550 555 560 Val Lys Glu Ala Leu Thr Ala Pro Ala Glu Gln Trp Ala Asn Asp Val 565 570 575 Glu Asp Asn Ala Glu Tyr Leu Thr Phe Leu Gly Pro Arg Val Ala 580 585 590 Glu Val His Ser Gln Phe Asp Ala Leu Lys Ala Arg Ile Ser Ala Ala 595 600 605 His Ala 610 <210> 3 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Phosphoenolpyruvate carboxykinase 52-G5 <400> 3 Met Thr Thr Ala Ala Ile Arg Gly Leu Gln Gly Glu Ala Pro Thr Lys 1 5 10 15 Asn Lys Glu Leu Leu Asn Trp Ile Ala Asp Ala Val Glu Leu Phe Gln 20 25 30 Pro Glu Ala Val Val Phe Val Asp Gly Ser Gln Ala Glu Trp Asp Arg 35 40 45 Met Ala Glu Asp Leu Val Glu Ala Gly Thr Leu Ile Lys Leu Asn Glu 50 55 60 Glu Lys Arg Pro Asn Ser Tyr Leu Ala Arg Ser Asn Pro Ser Serp Val 65 70 75 80 Ala Arg Val Glu Ser Arg Thr Phe Ile Cys Ser Glu Lys Glu Glu Asp 85 90 95 Ala Gly Pro Thr Asn Asn Trp Ala Pro Pro Gln Ala Met Lys Asp Glu 100 105 110 Met Ser Ser Ile Thr Leu Val Pro 115 120 <210> 4 <211> 702 <212> DNA <213> Corynebacterium glutamicum ATCC 13032 <220> <221> gene ≪ 222 > (1) <223> lysE <400> 4 atggaaatct tcattacagg tctgcttttg ggggccagtc ttttgctgtc catcggaccg 60 cagaatgtac tggtgattaa acaaggaatt aagcgcgaag gactcattgc ggttcttctc 120 gtgtgtttaa tttctgacgt ctttttgttc atcgccggca ccttgggcgt tgatcttttg 180 tccaatgccg cgccgatcgt gctcgatatt atgcgctggg gtggcatcgc ttacctgtta 240 tggtttgccg tcatggcagc gaaagacgcc atgacaaaca aggtggaagc gccacagatc 300 attgaagaaa cagaaccaac cgtgcccgat gacacgcctt tgggcggttc ggcggtggcc 360 actgacacgc gcaaccgggt gcgggtggag gtgagcgtcg ataagcagcg ggtttgggtg 420 aagcccatgt tgatggcaat cgtgctgacc tggttgaacc cgaatgcgta tttggacgcg 480 tttgtgttta tcggcggcgt cggcgcgcaa tacggcgaca ccggacggtg gattttcgcc 540 gctggcgcgt tcgcggcaag cctgatctgg ttcccgctgg tgggtttcgg cgcagcagca 600 ttgtcacgcc cgctgtccag ccccaaggtg tggcgctgga tcaacgtcgt cgtggcagtt 660 gtgatgaccg cattggccat caaactgatg ttgatgggtt ag 702 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> T7 promoter primer <400> 5 taatacgact cactataggg 20 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for lysE <400> 6 gctagttatt gctcagcgg 19 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for lysE <400> 7 gtcctaggtt aagtacttcc atag 24 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for lysE <400> 8 cctaggctaa cccatcaaca tcag 24
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