WO2014148743A1 - 퓨트레신 생산 재조합 미생물 및 이를 이용한 퓨트레신 생산방법 - Google Patents
퓨트레신 생산 재조합 미생물 및 이를 이용한 퓨트레신 생산방법 Download PDFInfo
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Definitions
- the present invention relates to a recombinant microorganism having improved putrescine productivity and a method for producing putrescine in high yield using the same.
- Polyamines such as spermidine and spermine, are present in most living cells, and putrescine or 1,4-butanediamine is a precursor to these spermidine and spermine metabolism. Used. Putrescine is found in gram-negative bacteria and fungi, and is present in high concentrations in various species and is expected to play an important role in the metabolism of microorganisms.
- putrescine is an important raw material for synthesizing polyamine nylon-4,6 by reacting with adipic acid, mainly acrylonitrile and succinonitrile from propylene.
- the fermentation is produced by chemical synthesis.
- This chemical synthesis is a three-step process that includes catalytic oxidation, cyanide compounds, and hydrogenation with high-pressure hydrogen, which is energy-consuming, environmentally friendly, and depleted of petroleum resources. There is a problem. Therefore, there is a demand for a method using biomass that is more environmentally friendly and can reduce energy consumption for the production of putrescine.
- WO09 / 125924 discloses ornithine biosynthetic pathways instead of inactivating the pathways involved in the degradation and utilization of putrescine present in E. coli, and inactivating the pathways in which ornithine, the precursor of putrescine, is converted to arginine.
- a method for producing putrescine in high yield is disclosed.
- Schneider (2010) discloses a method for producing high concentrations of putrescine by introducing a protein converting ornithine into putrescine into a strain of genus Corynebacterium having no putrescine producing ability and enhancing its activity. .
- E. coli putresin is introduced by a total of four pathways, which are introduced into cells by potABCD or potFGHI using ATP hydrolysis and by puuP of the H + symporter potE and puu pathway.
- PotFGHI is 0.5 mM
- potABCD is 1.5 mM
- potE is 1.8 mM
- puuP is 3.7 mM. It is considered to be.
- the potE transporter has both putrescine inlet and outlet functions.
- putresine enters the cell with protons.
- the pH is changed to acidic condition, it is expressed with the putrescine synthase speF and the extracellular ornithine is introduced into the cell and the putrescine synthesized in the cell is discharged to the cell (Kurihara et.al., J. Bacteriology 191: 8, 2776-2782, 2009).
- TPO1 and TPO4 are known which have high similarity to the amino acid sequence of Blt, a multidrug transporter of Bacillus.
- the two excreted proteins have the same characteristics as E. coli potE.
- putrescine, spermidine, and spermine are introduced into cells, while in acidic conditions, they have the function of excreting them extracellularly.
- TPO5 located in golgi or post-golgi secretory vesicles, exhibits resistance to 120 mM putrescine upon potentiation of expression, while susceptibility to 90 mM putrescine in defects (Tachihara et. Al, J. Biological Chemistry, 280 (13): 12637-12642, 2005).
- NCgl2522 functions as an excretion protein of putrescine in microorganisms of the genus Corynebacterium, a putrescine producing strain. It was confirmed that when the activity of NCgl2522 compared to the intrinsic activity, putrescine can be produced in high yield. In addition, when the expression of NCgl2522 in E. coli having a putrescine production pathway, the amount of putrescine in the culture medium was increased, confirming that NCgl2522 acts as a putrescine releasing protein in Escherichia coli, thus completing the present invention.
- One object of the present invention is to provide a recombinant microorganism which can be mutated to enhance NCgl2522 activity to produce putrescine in high yield.
- Another object of the present invention is to provide a method for producing putrescine in high yield using the microorganism.
- the present invention provides a microorganism having a putrescine production capacity, modified to enhance the activity of the protein comprising the amino acid sequence set forth in SEQ ID NO: 21 or 23.
- the invention is modified so that the activity of the microorganism is further involved in ornithine carbamoyl transferase (ArgF) and protein (NCgl1221) involved in glutamate release, compared to the intrinsic activity, ornithine dica
- ArgF ornithine carbamoyl transferase
- NCgl1221 protein involved in glutamate release
- a microorganism having a putrescine-producing ability in which the activity of a cyclase (ODC) is introduced.
- the present invention provides a protein (NCgl1221) wherein the ornithine carbamoyl transferase (ArgF) comprises the amino acid sequence of SEQ ID NO: 29, and is involved in glutamate release (NCgl1221). It provides an microorganism having a putrescine-producing ability, comprising an amino acid sequence described in, wherein ornithine dicarboxylase (ODC) comprises an amino acid sequence set forth in SEQ ID NO: 33.
- ornithine dicarboxylase ODC
- the present invention further provides that the microorganism further comprises acetyl gamma glutamyl phosphate reductase (ArgC), acetylglutamate synthase or ornithine acetyltransferase (ArgJ), acetylglutamate kinase (ArgB), and It provides a microorganism having putrescine production capacity, wherein the activity of acetylornithine aminotransferase (ArgD) is modified to be enhanced relative to intrinsic activity.
- the microorganism further comprises acetyl gamma glutamyl phosphate reductase (ArgC), acetylglutamate synthase or ornithine acetyltransferase (ArgJ), acetylglutamate kinase (ArgB), and It provides a microorganism having putrescine production capacity, wherein the activity of acetylornithine aminotrans
- the present invention provides the acetyl gamma glutamyl phosphate reductase (ArgC), acetylglutamate synthase or ornithine acetyltransferase (ArgJ), acetylglutamate kinase (ArgB), and acetylornithine aminotransfer.
- ArgC acetyl gamma glutamyl phosphate reductase
- ArgJ acetylglutamate synthase or ornithine acetyltransferase
- ArgB acetylglutamate kinase
- acetylornithine aminotransfer acetyl gamma glutamyl phosphate reductase
- ArgJ acetylglutamate synthase or ornithine acetyltransferase
- ArgB acetylglutamate kina
- the present invention provides a microorganism having a putrescine-producing ability, wherein the microorganism is additionally attenuated the activity of acetyltransferase.
- the present invention provides a microorganism having putrescine-producing ability, wherein the acetyltransferase comprises the amino acid sequence set forth in SEQ ID NO: 31 or 32.
- the present invention provides a microorganism having a putrescine-producing ability, wherein the microorganism is Escherichia microorganism or coryneform microorganism.
- the present invention provides a microorganism having putrescine-producing ability, wherein the microorganism is Escherichia coli or Corynebacterium glutamicum.
- the present invention provides a method for producing putrescine comprising culturing the microorganism having the putrescine production capacity to obtain a culture, and recovering putrescine from the cultured microorganism or the culture. .
- the present invention is modified so that NCgl2522 activity is enhanced in the corynebacterium genus microorganism having a putrescine-producing ability compared to the intrinsic activity to provide a recombinant corynebacterium microorganism with improved putrescine productivity.
- NCgl2522 refers to a permease belonging to the major facilitator superfamily (MFS) as the membrane protein identified in Corynebacterium glutamicum ATCC13032.
- MFS major facilitator superfamily
- NCgl2522 is known to excrete diaminopentane extracellularly from Corynebacterium glutamicum.
- the NCgl2522 activity is modified to be enhanced compared to endogenous activity, thereby increasing the release of putrescine produced in the cell, thereby providing a recombinant microorganism exhibiting high yield of putrescine productivity.
- intrinsic activity refers to the active state of an enzyme that is originally in an unmodified state, and "modified to be enhanced compared to the intrinsic activity” means that the enzyme activity of the pre-modification state is comparable. When the activity is newly introduced or further improved.
- enhancing enzyme activity includes not only the introduction of new enzymes or amplification of the activity of the enzyme itself, which induces abnormal effects, but also an increase in endogenous gene activity, endogenous gene amplification from internal or external factors, and Its activity is increased by deletion of inhibitory regulatory factors of gene expression, increase in gene copy number, introduction of genes from outside, modification of expression control sequences, in particular promoter replacement or modification, and increase in enzyme activity by intragenic mutations. do.
- modified to be enhanced compared to intrinsic activity means that a gene exhibiting activity is introduced or an increase in the number of copies of the gene, a deletion of an inhibitory regulatory factor of the gene expression, or a modification of an expression control sequence, for example, an improvement. It refers to a state in which the activity of the microorganisms after the manipulation is increased compared to the activity of the microorganisms before the manipulation such as the use of a promoter.
- NCgl2522 whose activity is increased by the present invention, is not particularly limited to, but is not limited to, a protein comprising the amino acid sequence of SEQ ID NO: 21 or 23, or at least 70%, preferably at least 80%, more preferably At least 90%, even more preferably at least 95%, even more preferably at least 98%, most preferably at least 99% homologous.
- a difference exists in the amino acid sequence of the protein showing the above activity depending on the species or strain of the microorganism but not limited thereto.
- “several” is different depending on the position and type in the three-dimensional structure of the amino acid residues of the protein, but specifically 2 to 20, preferably 2 to 10, more preferably 2 to 5 .
- Subsequent substitutions, deletions, insertions, additions, or inversions of these amino acids include those caused by naturally occurring mutations or artificial variations based on differences in individuals or species of microorganisms containing the activity of the polypeptide. do.
- Microorganisms in Corynebacterium do not have a putrescine biosynthetic pathway, but when ornithine decarboxylase (ODC) is introduced from the outside, putrescine is synthesized and putrescine is released into the cell. This indicates that there is an exiting protein, a transporter, acting as a putrescine pathway among numerous membrane proteins of the microorganism of the genus Corynebacterium.
- ODC ornithine decarboxylase
- NCgl2522 from Corynebacterium glutamicum ATCC13032 as a putrescine releasing protein comprises an amino acid sequence as set out in SEQ ID NO: 21 and has Corynebacterium glutamicum having 98% homology with the amino acid sequence.
- NCgl2522 from ATCC13869 comprises the amino acid sequence set forth in SEQ ID NO: 23.
- the polynucleotide encoding NCgl2522 of the present invention is at least 70%, preferably at least 80%, more preferably at least 90% of the amino acid sequence of SEQ ID NO: 21 or 23, or as long as it has similar activity as the NCgl2522 protein. Or more, even more preferably at least 95%, even more preferably at least 98%, most preferably at least 99%, and may comprise a polynucleotide encoding a protein, most preferably SEQ ID NO: 20 Or 22 nucleotide sequences.
- homology refers to identity between two amino acid sequences and is well known to those skilled in the art, using BLAST 2.0 to calculate parameters such as score, identity, similarity, and the like. It can be determined by the method.
- polynucleotide encoding NCgl2522 of the present invention may hybridize under stringent conditions with a nucleotide sequence of SEQ ID NO: 20 or 22 or a probe derived from the nucleotide sequence, and may function as a normally functioning NCgl2522.
- the coding may be variant.
- stringent conditions refers to conditions that enable specific hybridization between polynucleotides. For example, such stringent conditions can be found in J.
- modifying the activity of NCgl2522 to be enhanced compared to the intrinsic activity means that the copy number of the polynucleotide encoding the protein is increased, the expression control sequence is modified to increase the expression of the polynucleotide, and the activity of the enzyme is increased. Alteration of said polynucleotide sequence on a chromosome to be enriched, deletion of inhibitory regulatory factors of said gene expression, or a combination thereof.
- the increase in the number of copies of the polynucleotide is not particularly limited thereto, but may be performed in a form operably linked to a vector or by insertion into a chromosome in a host cell.
- a host capable of being cloned and functioning independently of a host, to which a polynucleotide encoding a protein of the present invention is operably linked may be performed by introducing into a host cell, or the host is operably linked to the polynucleotide.
- a vector capable of inserting the polynucleotide into the chromosome in the cell may be introduced into the host cell to increase the copy number of the polynucleotide in the chromosome of the host cell.
- the term "vector” refers to a DNA preparation containing the nucleotide sequence of the polynucleotide encoding said target protein operably linked to a suitable regulatory sequence so that it can express the target protein in a suitable host.
- the regulatory sequence includes a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating termination of transcription and translation. After being transformed into a suitable host cell, the vector can be replicated or function independent of the host genome and integrated into the genome itself.
- the vector to be used in the present invention is not particularly limited as long as it can replicate in a host cell, and any vector known in the art may be used.
- Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages.
- pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used as a phage vector or cosmid vector, and pBR-based, pUC-based, pBluescriptII-based, etc. , pGEM-based, pTZ-based, pCL-based and pET-based and the like can be used.
- the vector usable in the present invention is not particularly limited and known expression vectors can be used.
- pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector and the like can be used, and more preferably a pDZ vector can be used.
- a vector for inserting a chromosome in a bacterium may replace a polynucleotide encoding a target protein in a chromosome with a mutated polynucleotide. Insertion of the polynucleotide into the chromosome can be by any method known in the art, for example by homologous recombination. Since the vector of the present invention may be inserted into a chromosome by causing homologous recombination, the vector may further include a selection marker for confirming whether the chromosome is inserted.
- Selection markers are used to select cells transformed with a vector, i.e., to confirm the insertion of a polynucleotide of interest, and confer a selectable phenotype such as drug resistance, nutritional requirements, resistance to cytotoxic agents or expression of surface proteins. Markers can be used. In an environment in which a selective agent is treated, only cells expressing a selection marker survive or exhibit different expression traits, so that transformed cells can be selected.
- transformation in the present invention means introducing a vector comprising a polynucleotide encoding a target protein into a host cell to allow the protein encoded by the polynucleotide to be expressed in the host cell.
- Transformed polynucleotides include all of them, as long as they can be expressed in the host cell, whether they are inserted into or located outside the chromosome of the host cell.
- the polynucleotide also includes DNA and RNA encoding the target protein.
- the polynucleotide may be introduced in any form as long as it can be expressed by being introduced into a host cell.
- the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct containing all elements necessary for self expression.
- the expression cassette typically includes a promoter, transcription termination signal, ribosomal binding site and translation termination signal operably linked to the polynucleotide.
- the expression cassette may be in the form of an expression vector capable of self replication.
- the polynucleotide may be introduced into the host cell in its own form and operably linked with a sequence required for expression in the host cell.
- operably linked means that the gene sequence and the promoter sequence for initiating and mediating the transcription of the polynucleotide encoding the protein of interest of the present invention.
- modifying the expression control sequence to increase the expression of the polynucleotide is not particularly limited, but deletion, insertion, non-conservative or conservative substitution or their nucleic acid sequence to further enhance the activity of the expression control sequence. It can be carried out by inducing a variation in sequence in combination or by replacing with a nucleic acid sequence having stronger activity.
- the expression control sequences include, but are not limited to, promoters, operator sequences, sequences encoding ribosomal binding sites, sequences that control the termination of transcription and translation, and the like.
- a strong heterologous promoter may be linked to the top of the polynucleotide expression unit instead of the original promoter.
- the strong promoter include a CJ7 promoter, a lysCP1 promoter, an EF-Tu promoter, a groEL promoter, an aceA or aceB promoter, and the like.
- the lysCP1 promoter or the CJ7 promoter which is a Corynebacterium-derived promoter, may be operably linked to improve the expression rate of the polynucleotide encoding the enzyme.
- the lysCP1 promoter is an improved promoter through nucleotide sequence substitution of the polynucleotides encoding aspartate kinase and aspartate semialdehyde dehydrogenase. It is a powerful promoter that can be increased five times (WO 2009/096689).
- the CJ7 promoter can be expressed in Corynebacterium ammonia genes and Escherichia while searching for a region having a strong promoter sequence from Corynebacterium ammonia genes and as a promoter confirmed to have strong promoter activity.
- Nebacterium glutamicum is also a promoter of high intensity expression (Korean Patent Nos. 10-620092 and WO 2006/065095).
- modification of the polynucleotide sequence on the chromosome is not particularly limited, but the mutation in the expression control sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to further enhance the activity of the polynucleotide sequence. Or by replacing with a polynucleotide sequence that has been modified to have stronger activity.
- a polynucleotide having a nucleotide sequence of SEQ ID NO: 20 or 22 encoding NCgl2522 involved in putrescine release to provide a microorganism of the genus Corynebacterium with improved putrescine productivity is provided. It can be introduced into the gene to increase the number of copies, or to replace the NCgl2522 itself promoter with a promoter showing improved activity, preferably the CJ7 promoter having a nucleotide sequence of SEQ ID NO: 24.
- microorganism having a putrescine-producing ability or "microorganism producing a putrescine” means a microorganism to which putrescine-producing ability is imparted to a parent strain having no production capacity of putrescine.
- the microorganism to which the putrescine-producing ability or the putrescine is produced is not particularly limited, but the acetylglutamate synta converts glutamate to acetylglutamate to enhance the biosynthetic pathway from glutamate to ornithine.
- Ornithine acetyltransferase (ArgJ), which converts an acetyl ornithine to ornithine, acetylglutamate kinase (ArgB), which converts acetylglutamate to acetylglutamyl phosphate, and acetylglutamyl phosphate
- Acetyl gamma glutamyl phosphate reductase (ArgC) to convert glutamate semialdehyde
- acetyl ornithine aminotransferase to convert acetylglutamate semialdehyde to N-acetylornithine
- productivity of ornithine which is modified to increase the activity of D) relative to the intrinsic activity and used as a biosynthetic raw material of putrescine, may be improved.
- the microorganism may be ornithine carbamoyltransfrase (ArgF) involved in arginine synthesis in ornithine, a protein involved in the release of glutamate (NCgl1221), and / or a protein that acetylates putrescine (NCgl469). ) May be mutated to attenuate the activity of intrinsic activity and / or modified to introduce the activity of ornithine decarboxylase (ODC).
- ArgF ornithine carbamoyltransfrase
- ODC ornithine decarboxylase
- the acetyl gamma glutamyl phosphate reductase (ArgC), acetyl glutamate synthase or ornithine acetyl transferase (ArgJ), acetyl glutamate kinase (ArgB), acetyl ornithine aminotransferase (ArgD), ornithine Carbamoyl transferase (ArgF), protein involved in the release of glutamate (NCgl1221) and ornithine decarboxylase (ODC) are not particularly limited, but are preferably SEQ ID NOs: 25, 26, Amino acid sequences set forth in 27, 28, 29, 30, and 33 or amino acids having at least 70%, more preferably at least 80%, more preferably at least 90% homology thereof.
- the protein for acetylating putrescine (NCgl469) is not particularly limited thereto, but is preferably an amino acid sequence of SEQ ID NO: 31 or 32 or 70% or more thereof, more preferably 80% or more, more preferably May comprise an amino acid sequence having at least 90% homology.
- acetyl gamma glutamyl phosphate reductase (ArgC), acetylglutamate synthase or ornithine acetyltransferase (ArgJ), acetylglutamate kinase (ArgB), acetylornithine aminotransferase (ArgD) and ornithine
- Increasing the activity of the decarboxylase can be controlled by the methods described above in connection with the increased activity of NCgl2522, such as an increase in the number of copies of the polynucleotide encoding the protein and an increase in the expression of the polynucleotide. Modification of the sequence, modification of the polynucleotide sequence on a chromosome to enhance the activity of the enzyme, deletion of a regulator that inhibits expression of the polynucleotide of the enzyme, and combinations thereof.
- the active weakening of the protein involved in the release of ornithine carbamoyl transferase (ArgF) and glutamate (NCgl1221), and the protein acetylating putrescine (NCgl469) may be a part of a polynucleotide encoding the protein or Deletion in whole, modification of expression control sequences to reduce expression of the polynucleotide, modification of the polynucleotide sequence on a chromosome to attenuate the activity of the protein, and combinations thereof may be achieved.
- a method of deleting part or all of a polynucleotide encoding a protein may include a polynucleotide encoding a target protein inherent in a chromosome through a bacterial chromosome insertion vector, into a polynucleotide or a marker gene in which some nucleic acid sequences are deleted. By replacement.
- the term "some” differs depending on the kind of polynucleotide, but is specifically 1 to 300, preferably 1 to 100, more preferably 1 to 50.
- the method of modifying the expression control sequence is carried out by inducing a mutation on the expression control sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof to further weaken the activity of the expression control sequence, or more By replacing with a nucleic acid sequence having weak activity.
- the expression control sequence includes a promoter, an operator sequence, a sequence encoding a ribosomal binding site, and a sequence that controls the termination of transcription and translation.
- a method of modifying a polynucleotide sequence on a chromosome is carried out by inducing a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof to further weaken the activity of the enzyme, or weaker. By replacing with a polynucleotide sequence that has been improved to have activity.
- the method of deleting a regulator that inhibits the expression of the polynucleotide of the enzyme can be carried out by replacing the polynucleotide of the expression inhibitory factor with a polynucleotide or marker gene which is missing some nucleic acid sequence.
- the term "some" differs depending on the kind of polynucleotide, but is specifically 1 to 300, preferably 1 to 100, more preferably 1 to 50.
- the microorganism of the present invention is a microorganism having a putrescine-producing ability, and includes a prokaryotic microorganism in which a protein including the amino acid sequence of SEQ ID NO: 21 or 23 is expressed, and examples thereof include Escherichia spp. Escherichia sp., Shigella sp., Citrobacter sp., Salmonella sp., Enterobacter sp. Yersinia sp. Klebsiella sp., Erwinia sp., Corynebacterium sp., Brevibacterium sp., Lactobacillus sp.
- the microorganism of the present invention is a microorganism belonging to the genus Escherichia or a microorganism belonging to the genus Corynebacterium, and more preferably E. coli or Corynebacterium glutamicum .
- the microorganism of the genus Corynebacterium of Accession No. KCCM11138P (Korean Patent Publication No. 2012-0064046)
- a microorganism (Korean Patent Application No. 2012-0003634) belonging to the genus Corynebacterium of Accession No. KCCM11240P was used.
- putrescine based on Corynebacterium glutamicum ATCC13869 each having the same genotype as KCCM11138P and KCCM11240P, both of the Corynebacterium glutamicum ATCC13032-based putrescine producing strains.
- Production strains DAB12-a and DAB12-b were used.
- the ATCC13869 strain can be obtained from the American Type Culture Collection (ATCC). That is, each strain is assigned a unique registration number listed in the catalog of the ATCC and can be ordered using this registration number.
- the putrescine producing strain DAB12-a is missing a gene encoding ornithine carbamoyl transferase (ArgF) from Corynebacterium glutamicum ATCC13869 and a gene encoding glutamate excretion protein NCgl1221, orni Gene encoding chitin decarboxylase (OCD) is introduced, characterized in that the promoter of ornithine biosynthetic gene operon (argCJBD) is substituted with an improved promoter.
- putrescine-producing strain DAB12-b is characterized in that the activity of protein NCgl1469, which acetylates putrescine in the DAB12-a strain, is modified to be weaker than intrinsic activity.
- a gene encoding ornithine carbamoyl transferase (ArgF) in the chromosome and a protein involved in glutamate release (NCgl1221) in wild-type Corynebacterium glutamicum ATCC13032 The gene is deleted, the ArgCJBD family of genes encoding enzymes involved in ornithine synthesis in glutamate are replaced with an improved promoter, and the gene encoding ornithine decarboxylase (ODC) is introduced into the chromosome.
- ArgF ornithine carbamoyl transferase
- Corynebacterium glutamicum KCCM11138P and Corynebacterium glutamicum KCCM11240P which attenuated the gene encoding the acetyltransferase NCgl1469 in the microorganism, were prepared as putrescine-producing strains.
- NCgl2522-deficient strains derived from Corynebacterium glutamicum ATCC13032 plasmid pDZ-1'NCgl2522 (K / O) was prepared based on the base sequence of NCgl2522 derived from Corynebacterium glutamicum ATCC13032. It was.
- the plasmid pDZ-1′NCgl2522 (K / O) was transduced into the prepared putrescine producing strains KCCM11138P and KCCM11240P, respectively, and were selected as NCgl2522 deficient strains, and these strains were named KCCM11138P ⁇ NCgl2522 and KCCM11240P ⁇ NCgl2522, respectively.
- NCgl2522-deficient strains derived from Corynebacterium glutamicum ATCC13869 were prepared and named DAB12-a ⁇ NCgl2522 and DAB12-b ⁇ NCgl2522.
- the CJ7 promoter (KCCM10617, Korean Registered Patent) which newly introduces the NCgl2522 in the transposon of the Corynebacterium glutamicum strain or the NCgl2522 itself promoter in the chromosome is newly developed by the applicant. 10-0620092).
- the present inventors have modified the activity of NCgl2522 in putrescine-producing strain Corynebacterium glutamicum KCCM11138P to be enhanced compared to the intrinsic activity to show increased putrescine excretion ability, thereby improving putrescine productivity.
- the microorganism of the genus was named Corynebacterium glutamicum CC01-0510, and was deposited with the Korean Culture Center of Microorganisms (KCCM) on March 8, 2013 under the Budapest Treaty and was given accession number KCCM11401P.
- the step of culturing the microorganism is not particularly limited thereto, but is preferably performed by a known batch culture method, continuous culture method, fed-batch culture method or the like.
- the culture conditions are not particularly limited, but using a basic compound (e.g. sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound (e.g. phosphoric acid or sulfuric acid) to an appropriate pH (e.g. pH 5 to 9, preferably pH 6 to 8, most preferably pH 6.8) can be adjusted, and the aerobic conditions can be maintained by introducing oxygen or oxygen-containing gas mixture into the culture, the culture temperature is 20 to 45 °C, preferably 25 to 40 ° C. can be maintained and incubated for about 10 to 160 hours. Putrescine produced by the culture may be secreted into the medium or remain in the cells.
- a basic compound e.g. sodium hydroxide, potassium hydroxide or ammonia
- an acidic compound e.g. phosphoric acid or sulfuric acid
- pH e
- the culture medium used may include sugars and carbohydrates (e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), fats and fats (e.g. soybean oil, sunflower seeds) as carbon sources.
- sugars and carbohydrates e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose
- fats and fats e.g. soybean oil, sunflower seeds
- Oils, peanut oils and coconut oils fatty acids (e.g. palmitic acid, stearic acid and linoleic acid), alcohols (e.g. glycerol and ethanol) and organic acids (e.g. acetic acid), etc.
- Nitrogen sources include nitrogen-containing organic compounds such as peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean meal and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and Ammonium nitrate) and the like can be used individually or in combination;
- As a source of phosphorus, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, a corresponding sodium-containing salt, and the like can be used individually or in combination;
- Other metal salts such as magnesium sulfate or iron sulfate, and essential growth-promoting substances such as amino acids and vitamins.
- the method for recovering putrescine produced in the culturing step of the present invention collects the desired amino acid from the culture medium using a suitable method known in the art according to a culture method, for example, a batch, continuous or fed-batch culture method. can do.
- the microorganism of the genus Corynebacterium improved productivity of putrescine of the present invention was modified to enhance the activity of NCgl2522, which releases putrescine in cells, rather than endogenous activity, thereby increasing the release of putrescine into the cell, It was also confirmed that the resistance to.
- NCgl2522 derived from Corynebacterium glutamicum can be widely used for the production of effective putrescine by applying to microorganisms having putrescine production ability.
- FIG. 1 is a schematic diagram showing that NCgl2522 is included in a clone (B19) finally selected from a transformed colony in which a Corynebacterium chromosome library is introduced according to the present invention.
- NCgl2522 is deleted or enhanced according to the present invention.
- a gene encoding ornithine decarboxylase (ODC) derived from wild type Escherichia coli W3110, which is involved in the synthesis of putrescine in ornithine was introduced into the strain.
- Corynebacterium glutamicum strain having putrescine-producing ability was prepared by substituting the promoter of the argCJBD gene group, which encodes an enzyme involved in ornithine synthesis in glutamate, with the CJ7 promoter, an improved promoter.
- the argCJBD is acetyl gamma glutamyl phosphate reductase (ArgC), acetyl glutamate synthase or ornithine acetyltransferase (ArgJ), acetylglutamate kinase (ArgB), acetylornithine involved in the ornithine biosynthesis pathway in glutamate It encodes a tin aminotransferase (ArgD).
- the Corynebacterium glutamicum strain having the putrescine-producing ability thus produced was deposited on November 24, 2010 to the Korea Microorganism Conservation Center (KCCM) under the Budapest Treaty, and was given accession number KCCM11138P. Detailed procedures relating to the production of microorganisms of the genus Corynebacterium having the ability to produce putrescine are described in detail in Korean Patent Publication No. 2012-0064046, which is hereby incorporated by reference in its entirety.
- N-acetyl purine is weakened by attenuating the gene encoding NCgl1469, an acetyltransferase, in the Corynebacterium glutamicum KCCM11138P prepared in Reference Example 1. Corynebacterium glutamicum strains with increased putrescine production were produced by not producing trecine.
- primer pairs of SEQ ID NO: 1 and 2 for obtaining homologous recombination fragments of the N-terminal region of NCgl1469, and C of NCgl1469 were prepared as shown in Table 1 below.
- NCgl1469-del-F1_BamHI SEQ ID NO: 1
- CGGGATCCAACCTTCAGAACGCGAATAC NCgl1469-del-R1_SalI
- CGCGTCGACTTGGCACTGTGATTACCATC NCgl1469-del-F2_SalI
- SEQ ID NO: 3 CGCGTCGACTTGGGTTATATCCCCTCAGA NCgl1469-del-R2_XbaI (SEQ ID NO: 4) TGCTCTAGATAGTGAGCCAAGACATGGAA
- PCR was performed using two pairs of primers to obtain PCR fragments of the N-terminal site and the C-terminal site, respectively, followed by electrophoresis. The desired fragment was obtained. At this time, the PCR reaction was repeated 30 times of denaturation at 95 °C, 30 seconds annealing at 55 °C and 30 seconds elongation process at 72 °C.
- the fragments obtained from the N-terminal region were treated with restriction enzymes BamHI and SalI, and the fragments of the C-terminal region with restriction enzymes SalI and XbaI, and the treated fragments were treated with pDZ vectors treated with restriction enzymes BamHI and XbaI. Cloning to make plasmid pDZ-NCgl1469 (K / O).
- the plasmid pDZ-NCgl1469 (K / O) was introduced into Corynebacterium glutamicum KCCM11138P by electroporation to obtain a transformant, and the transformant was kanamycin (25 ⁇ g / ml) and X-gal ( Colonies were formed by smearing and incubating in BHIS plate medium containing 5-bromo-4-chloro-3-indolin--D-galactoside (Braine heart infusion 37 g / l, sorbitol 91 g / l, agar 2%) I was.
- a strain in which the plasmid pDZ-NCgl1469 (K / O) was introduced was selected by selecting a blue colony from the colonies formed therefrom.
- CM medium glucose medium
- yeast extract 5 g / l yeast extract 5 g / l
- beef extract 5 g / l NaCl 2.5 g / l, urea 2 g / l, pH 6.8
- Inoculation was incubated for 8 hours at 30 ° C shaking, and diluted sequentially from 10 -4 to 10 -10 , respectively, plated and cultured in X-gal-containing solid medium to form colonies.
- white colonies appearing at a relatively low rate were selected to generate a Corynebacterium glutamicum strain having improved productivity of putrescine due to the deletion of the gene encoding NCgl1469.
- the Corynebacterium glutamicum strain with the improved ability to produce putrescine was named as KCCM11138P NCgl1469, and was deposited on December 26, 2011 to the Korea Microorganism Conservation Center (KCCM) under the Budapest Treaty, and gave accession number KCCM11240P. received.
- KCCM Microorganism Conservation Center
- Detailed procedures relating to the production of microorganisms of the genus Corynebacterium having a putrescine-producing ability are described in detail in Korean Patent Application No. 2012-0003634, which is incorporated herein by reference in its entirety.
- Corynebacterium glutamicum does not have a putrescine biosynthesis pathway, but when ornithine decarboxylase is introduced from the outside and putrescine is capable of synthesizing putrescine, putrescine is released from the cell. This indicates the presence of a transporter protein that acts as a putrescine outlet pathway among numerous membrane proteins of the microorganism of the genus Corynebacterium.
- a chromosome library of wild type Corynebacterium glutamicum ATCC13032 was prepared. Specifically, incomplete cleavage was performed by treating the chromosome of Corynebacterium glutamicum ATCC13032 with restriction enzyme Sau3AI, and a pECCG122 vector (E. coli and Corynebacterium) treated with BamHI by separating gene fragments of 3 to 5 kb in size.
- Shuttle vector of Korean Patent Publication No. 1992-00009331303 Korean Patent Publication No. 1992-0000933
- Corynebacterium chromosome library obtained therefrom was transformed into Corynebacterium glutamicum KCCM11138P, a putrescine producing strain according to 1, and then 0.35 M putrescine-containing minimal medium (based on 1 L of distilled water).
- NCgl2522 isolated from Corynebacterium glutamicum ATCC13032 as a putrescine excretion protein, has an amino acid sequence as set out in SEQ ID NO: 21, which is encoded from a polynucleotide having a nucleotide sequence as set out in SEQ ID NO: 20 .
- NCgl2522 from Corynebacterium glutamicum ATCC13032 was constructed to delete the gene encoding NCgl2522.
- primer pairs of SEQ ID NOs: 5 and 6 for obtaining homologous recombination fragments of the N-terminal region of NCgl2522 based on the nucleotide sequence of SEQ ID NO: 20 of the gene encoding NCgl2522, and C- of NCgl2522.
- Primer pairs of SEQ ID NOs: 7 and 8 to obtain homologous fragments of the terminal sites were prepared as shown in Table 2 below.
- NCgl2522-del-F1_BamHI SEQ ID NO: 5
- CGGGATCCCACGCCTGTCTGGTCGC NCgl2522-del-R1_SalI
- ACGCGTCGACGGATCGTAACTGTAACGAATGG NCgl2522-del-F2_SalI
- ACGCGTCGACCGCGTGCATCTTTGGACAC NCgl2522-del-R2_XbaI
- PCR using genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, PCR using two pairs of primers was performed to amplify PCR fragments of the N-terminal and C-terminal regions of the NCgl2522 gene, respectively. Electrophoresis gave the desired fragment. At this time, the PCR reaction was repeated 30 times of denaturation at 95 °C, 30 seconds annealing at 55 °C and 30 seconds elongation process at 72 °C.
- the fragments obtained from the N-terminal region were treated with restriction enzymes BamHI and SalI, and the fragments of the C-terminal region with restriction enzymes SalI and XbaI, and the treated fragments were treated with pDZ vectors treated with restriction enzymes BamHI and XbaI. Cloning to make plasmid pDZ-1'NCgl2522 (K / O).
- the plasmid pDZ-1'NCgl2522 (K / O) was introduced into Corynebacterium glutamicum KCCM11138P and KCCM11240P according to Reference Examples 1 and 2, respectively, by electroporation to obtain a transformant.
- the transformants were BHIS plate medium (Braine heart infusion 37 g / L, sorbitol 91 g) containing kanamycin (25 ⁇ g / ml) and X-gal (5-bromo-4-chloro-3-indolin--D-galactoside). / l, agar 2%) to form a colony by incubation.
- a strain in which the plasmid pDZ-1'NCgl2522 (K / O) was introduced was selected by selecting a blue colony from the colonies formed therefrom.
- CM medium glucose 10 g / l, polypeptone 10 g / l, yeast extract 5 g / l, beef extract 5 g / l, NaCl 2.5 g / l, urea 2 g / l, pH 6.8
- Shake culture (30 °C, 8 hours)
- 10 -4 to 10 -10 plated and cultured in X-gal-containing solid medium to form colonies.
- NCgl2522 was deleted, and the Corynebacterium glutamicum mutant KCCM11138P ⁇ NCgl2522 and KCCM11240P ⁇ , respectively. It was named NCgl2522.
- the genomic DNA of Corynebacterium glutamicum ATCC13869 was used as a template, and SEQ ID NOs: 5 and 8 PCR was performed using primer pairs. At this time, the PCR reaction was repeated 30 times of denaturation of 30 seconds at 95 °C, annealing of 30 seconds at 55 °C and extension of 2 minutes at 72 °C. PCR product obtained therefrom was subjected to sequencing, followed by sequencing.
- NCgl2522 derived from Corynebacterium glutamicum ATCC13869 includes the nucleotide sequence set forth in SEQ ID NO: 22. Proteins encoded from include amino acid sequences set forth in SEQ ID NO: 23. A comparison of the amino acid sequences of NCgl2522 from Corynebacterium glutamicum ATCC13032 and NCgl2522 from Corynebacterium glutamicum ATCC13869 confirmed that they had 98% sequence homology.
- the genomic DNA of Corynebacterium glutamicum ATCC13869 was used as a template, and 2 shown in Table 2 was used.
- PCR using each pair of primers was performed to amplify PCR fragments at the N-terminal and C-terminal regions of the NCgl2522 gene, respectively, and then electrophoresed to obtain the desired fragments.
- the PCR reaction was repeated 30 times of denaturation at 95 °C, 30 seconds annealing at 55 °C and 30 seconds elongation process at 72 °C.
- the fragments of the N-terminal site obtained therefrom were treated with restriction enzymes BamHI and SalI, and the fragments of the C-terminal site obtained were treated with restriction enzymes SalI and XbaI, and the treated fragments were treated with pDZ treated with restriction enzymes BamHI and XbaI.
- Cloning into a vector produced the plasmid pDZ-2'NCgl2522 (K / O).
- the plasmid pDZ-2'NCgl2522 (K / O) was transformed into Corynebacterium glutamicum DAB12-a and DAB12-b in the same manner as in Example ⁇ 2-1> to delete the gene encoding NCgl2522. Strains were selected. Corynebacterium glutamicum mutants selected therefrom were named DAB12-a ⁇ NCgl2522 and DAB12-b ⁇ NCgl2522, respectively.
- Corynebacterium glutamicum mutants (KCCM11138P ⁇ NCgl2522, KCCM11240P ⁇ NCgl2522, DAB12-a ⁇ NCgl2522, and DAB12-b ⁇ NCgl2522) and four parent strains (KCCM11138P, KCCM11240a, DAB12) , And DAB12-b) were prepared using 1 mM arginine CM plate medium (glucose 1%, polypeptone 1%, yeast extract 0.5%, beef extract 0.5%, NaCl 0.25%, urea 0.2%, 50% NaOH 100 ⁇ l, agar 2). %, pH 6.8, 1 L basis) and incubated for 24 hours at 30 °C.
- NCgl2522 was introduced into the transposon gene to confirm the effect of high putrescine production through the further insertion of the NCgl2522 gene (including its own promoter site) into the chromosome in the Corynebacterium microorganism KCCM11138P with putrescine production ability.
- a transgenic vector pDZTn (Korean Patent Publication No. 2008-0033054), which enables transduction of genes in a chromosome using a transposon gene region of a microorganism of Corynebacterium, was used.
- the NCgl2522 gene including the autologous promoter, amplified a gene fragment of about 1.88 kb using primer pairs of SEQ ID NOs: 9 and 10, using a chromosome of ATCC13032 strain as a template (see Table 4). At this time, the PCR reaction was repeated 30 times of denaturation of 30 seconds at 95 °C, annealing of 30 seconds at 55 °C and 30 seconds or 2 minutes elongation at 72 °C. The PCR product was electrophoresed on 0.8% agarose gel and then purified by eluting a band of desired size. The pDZTn vector was treated with XhoI and fusion cloned the NCgl2522 PCR product of ATCC13032 strain. Fusion cloning was performed using an In-FusionHD Cloning Kit (Clontech). The resulting plasmid was named pDZTn-1'NCgl2522.
- the plasmid pDZTn-1'NCgl2522 was introduced into Corynebacterium glutamicum KCCM11138P described in Reference Example 1 by electroporation to obtain a transformant, and the transformant was subjected to the same method as in Example 2. A strain in which NCgl2522 was introduced into the transposon was selected.
- Genomic DNA obtained from the selected strains was used as a template, and PCR was performed using primer pairs of SEQ ID NOs: 9 and 10 to confirm that NCgl2522 in the transposon was introduced by introduction of the plasmid pDZTn-1'NCgl2522. At this time, the PCR reaction was repeated 30 times at 30 °C denaturation at 94 °C, 30 seconds annealing at 55 °C and 2 minutes extension at 72 °C.
- Corynebacterium glutamicum mutants selected therefrom were named KCCM11138P Tn: 1′NCgl2522.
- CJ7 promoter (WO 2006/65095) was introduced before the NCgl2522 start codon in the chromosome.
- a homologous recombination fragment comprising a CJ7 promoter having the nucleotide sequence set forth in SEQ ID NO: 24, wherein both ends of the promoter had the original sequence of NCgl2522 on the chromosome.
- the 5'-terminal portion of the CJ7 promoter was obtained by performing genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and performing PCR using primer pairs of SEQ ID NOs: 11 and 12. At this time, the PCR reaction was repeated 30 times of denaturation at 94 °C, 30 seconds annealing at 55 °C and 30 seconds stretching at 72 °C.
- the CJ7 promoter site was obtained by performing PCR under the same conditions using primer pairs of SEQ ID NOs: 13 and 14, and the 3'-terminal site of the CJ7 promoter templated genomic DNA of Corynebacterium glutamicum ATCC13032. It was obtained by performing PCR under the same conditions using the primer pair of SEQ ID NO: 15 and 16. Primers used for promoter substitution are as shown in Table 5 below.
- NCgl2522-L5 (SEQ ID NO: 11) TGCAGGTCGACTCTAGAGTTCTGCGTAGCTGTGTGCC NCgl2522-L3 (SEQ ID NO: 12) GGATCGTAACTGTAACGAATGG CJ7-F (SEQ ID NO: 13) CGTTACAGTTACGATCCAGAAACATCCCAGCGCTACTAATA CJ7-R (SEQ ID NO .: 14) AGTGTTTCCTTTCGTTGGGTACG NCgl2522-R5 (SEQ ID NO .: 15) CAACGAAAGGAAACACTATGACTTCAGAAACCTTACAGGCG NCgl2522-R3 (SEQ ID NO .: 16) TCGGTACCCGGGGATCCCACAAAAAGCGTAGCGATCAACG
- PCR product obtained above was fusion cloned into pDZ vector treated with BamHI and XbaI. Fusion cloning was performed using an In-FusionHD Cloning Kit (Clontech). The resulting plasmid was named pDZ-P (CJ7) -1'NCgl2522.
- the plasmid pDZ-P (CJ7) -1′NCgl2522 prepared above was introduced into Corynebacterium glutamicum KCCM11138P and KCCM11240P according to Reference Examples 1 and 2, respectively, to prepare a transformant.
- the prepared transformants were inoculated in CM medium and shaken at 30 ° C. for 8 hours, and the obtained cultures were diluted to 10 ⁇ 4 to 10 ⁇ 10 to contain 25 ⁇ g / ml kanamycin and X-gal. Colonies were formed by plating and incubating in BHIS plate medium.
- strains were finally selected in which the NCgl2522 promoter was replaced by the CJ7 promoter by the second crossing.
- Genomic DNA obtained from the selected strain was used as a template, and PCR was carried out using primer pairs of SEQ ID NOs: 13 and 16 to introduce the CJ7 promoter in front of the NCgl2522 start codon in the chromosome by introduction of the plasmid pDZ-1'CJ7 (NCgl2522). It was confirmed that it was introduced.
- the PCR reaction was repeated 30 times with 30 seconds of denaturation at 94 ° C, 30 seconds of annealing at 55 ° C, and 1 minute extension at 72 ° C.
- Corynebacterium glutamicum mutants selected therefrom were named KCCM11138P P (CJ7) -NCgl2522 and KCCM11240P P (CJ7) -NCgl2522, respectively.
- NCgl2522 (including the promoter region) was determined to be introduced into the transposon gene NCgl2522
- the gene amplified a gene fragment of about 1.97 kb using primer pairs of SEQ ID NOs: 17 and 10, using the chromosome of the ATCC13869 strain as a template (see Table 6).
- the PCR reaction was repeated 30 times with 30 seconds of denaturation at 94 ° C., 30 seconds with annealing at 55 ° C., and 30 seconds or 2 minutes at 72 ° C.
- NCgl2522 PCR fragment was fusion cloned into the pDZTn vector treated with XhoI. Fusion cloning was performed using an In-FusionHD Cloning Kit (Clontech). The resulting plasmid was named pDZTn-2'NCgl2522.
- the plasmid pDZTn-2'NCgl2522 was transformed into Corynebacterium glutamicum DAB12-a in the same manner as in Example ⁇ 3-1> to confirm that NCgl2522 in the transposon was introduced.
- Corynebacterium glutamicum mutants selected therefrom were named DAB12-a Tn: 2'NCgl2522.
- the genomic DNA of Corynebacterium glutamicum ATCC13869 was used as a template to introduce the CJ7 promoter before the start codon of NCgl2522 derived from Corynebacterium glutamicum ATCC13869, and the following Table 7 PCR was performed using each of the three pairs of primers described in the above to amplify the PCR fragments of the CJ7 promoter site, its N-terminal site and C-terminal site, respectively, followed by electrophoresis to obtain the desired fragments. At this time, the PCR reaction was repeated 30 times of denaturation at 94 °C, 30 seconds annealing at 55 °C and 30 seconds stretching at 72 °C.
- PCR fragments of the CJ7 promoter site, its N-terminal site and C-terminal site obtained therefrom were fusion cloned into pDZ vectors treated with BamHI and XbaI. Fusion cloning was performed using an In-FusionHD Cloning Kit (Clontech). The resulting plasmid was named pDZ-P (CJ7) -2'NCgl2522.
- the plasmid pDZ-'P (CJ7) -2'NCgl2522 was transformed to Corynebacterium glutamicum DAB12-a and DAB12-b in the same manner as in Example ⁇ 3-2>, respectively, to the CJ7 promoter before the NCgl2522 initiation codon.
- the strain into which was introduced was selected.
- Corynebacterium glutamicum mutants selected therefrom were named DAB12-a P (CJ7) -NCgl2522 and DAB12-b P (CJ7) -NCgl2522, respectively.
- Corynebacterium glutamicum mutants KCCM11138P Tn 1'NCgl2522 and Intracellular putrescine concentration was measured in the parent strain KCCM11138P by the extraction method using an organic solvent. Intracellular metabolite analysis was performed according to the method described in Nakamura J et al., Appl. Environ. Microbiol. 73 (14): 4491-4498, 2007.
- Corynebacterium glutamicum mutant KCCM11138P Tn 1'NCgl2522 and the parent strain KCCM11138P were each added with 1 mM arginine CM liquid medium (glucose 1%, polypeptone 1%, yeast extract 0.5%, beef extract 0.5%, NaCl 0.25). %, urea 0.2%, 50% NaOH 100 l, pH 6.8, 1 L basis) inoculated in 25 ml and then incubated shaking at 30 °C 200 rpm. When cell growth reached an exponential phase during the culture, cells were separated from the culture via rapid vacuum filtration (Durapore HV, 0.45 m; Millipore, Billerica, Mass.).
- the cell-adsorbed filter was washed twice with 10 ml of cooling water and then soaked in methanol containing 5 M morpholine ethanesulfonic acid and 5 M methionine sulfone for 10 minutes. After the same amount of chloroform and 0.4 times the volume of the well mixed with the extracted solution, only the aqueous phase (aqueous phase) was applied to the spin column (spin column) to remove protein contaminants. The filtered extract was analyzed using capillary electrophoresis mass spectrometry, and the results are shown in Table 9 below.
- putrescine resistance was evaluated in KCCM11240P, KCCM11240P ⁇ NCgl2522, and KCCM11240P P (CJ7) -NCgl2522 strains prepared above.
- Each strain was inoculated in 2 ml CMA liquid medium and incubated at 30 ° C. for about 10 hours, and then diluted in the order of 10 5 , 10 4 , 10 3 , 10 2 and 10 1 .
- Each prepared dilution was carried out with a CMA plate medium containing 1M putrescine 0 M or 0.8 M (glucose 1%, polypeptone 1%, yeast extract 0.5%, beef extract 0.5%, NaCl 0.25%, urea 0.2%.
- Agar 1.8%, 1 The growth was compared between strains by spotting in mM arginine, pH 6.8, 1 L) and incubating for 48 hours at 30 ° C.
- the strains showed two different growth patterns. As shown in FIG. 2, the strains lacking the NCgl2522 gene did not grow under the high concentration of putrescine, whereas the expression of the NCgl2522 gene was enhanced. In the strain, cell growth increased under the same conditions. This result shows that the increase in putrescine excretion due to the enhancement of NCgl2522 gene confirms the high cell growth potential compared to the parent strain under conditions containing high concentrations of putrescine, indicating that the introduction and strengthening of NCgl2522 is essential for the high concentration of putrescine. will be.
- Example 6 NCgl2522 in Escherichia Coli Putrescine fermentation through introduction
- the W3110 chromosome was used as a template and amplified about 2.1 kb of the speC gene fragment using primer pairs of SEQ ID NOs: 34 and 35 (see Table 10).
- the PCR product was electrophoresed on 0.8% agarose gel and then purified by eluting a band of desired size.
- the pSE280 vector containing the Trc promoter (Invitrogen) was treated with NcoI and EcoRI followed by fusion cloning of the speC PCR product. Fusion cloning was performed using an In-Fusion® HD Cloning Kit (Clontech). The resulting plasmid was named pSE280-speC.
- PSE280 was used as a template for constructing the NCgl2522 expression vector, and a Trc promoter fragment was obtained using primer pairs of SEQ ID NOs: 36 and 37, and the Corynebacterium glutamicum ATCC13032 chromosome was used as the template, SEQ ID NOs: 38 and 39 NCgl2522 fragment was obtained using a primer pair of.
- the PCR product was electrophoresed on 0.8% agarose gel and then purified by eluting a band of desired size.
- the trc promoter fragment and the NCgl2522 fragment were fusion cloned into pcc1BAC treated with HindIII.
- the resulting plasmid was named pcc1BAC-P (trc) -NCgl2522.
- the plasmids pSE280-speC and pcc1BAC-P (trc) -NCgl2522 were transformed into W3110.
- E. coli transformation was carried out using 2 ⁇ TSS solution (Epicentre), and E. coli with pSE280-speC was used as LB plate medium (Tryptone 10 g, yeast extract 5 g, containing 100 ⁇ g / ml). Colonies were formed by plating and incubating in 10 g of Nacl, 2% agar, 1 L).
- Escherichia coli into which pcc1BAC-P (trc) -NCgl2522 was introduced formed colonies by plating on LB plate medium containing chloramphenicol (35 ⁇ g / ml). Putrescine production capacity was confirmed with the strains obtained above.
- W3110, W3110 pSE280-speC, and W3110 pcc1BAC-P (trc) -NCgl2522 were inoculated in LB, LA, and LC solid medium, respectively, and incubated at 37 ° C. for 24 hours, and each of 25 ml titer medium ((NH 4 ) 2 PO 4 2 g, KH 2 PO 4 6.75 g, citric acid 0.85 g, MgSO 4 7H 2 O 0.7 g, trace element 0.5% (v / v), glucose 10 g, AMS 3 g, CaCO 3 30 g , 1 L standard) was incubated for 24 hours at 37 °C.
- 25 ml titer medium ((NH 4 ) 2 PO 4 2 g, KH 2 PO 4 6.75 g, citric acid 0.85 g, MgSO 4 7H 2 O 0.7 g, trace element 0.5% (v / v), glucose 10 g, AMS 3 g, Ca
- Trace metal solution is 5 M HCl per liter: 10 g FeSO 4 ⁇ 7H 2 O 10 g, ZnSO 4 ⁇ 7H 2 O 2.25 g, CuSO 4 ⁇ 5H 2 O 1 g, MnSO 4 ⁇ 5H 2 O 0.5 g, 0.22 g Na 2 B 4 O 7 .10H 2 O, CaCl 2 .2H 2 O 2 g, and (NH 4 ) 6 Mo 7 O 2 .4H 2 O.
- the putrescine concentration produced from each culture was measured and the results are shown in Table 11 below.
- NCgl2522 gene is active in E. coli as a putrescine releasing protein.
- the present inventors introduced the NCgl2522 in the transposon from the microorganism KCCM11138P of the Corynebacterium genus having the putrescine-producing ability to enhance the NCgl2522 activity of the Corynebacterium glutamicum strain in high yield through the increased putrescine excretion capacity After confirming that the putrescine can be produced, the strain was named Corynebacterium glutamicum CC01-0510, and was deposited with the Korea Microorganism Conservation Center (KCCM) on March 08, 2013 under the Budapest Treaty to receive the accession number KCCM11401P.
- KCCM Korea Microorganism Conservation Center
- the microorganism of the genus Corynebacterium improved productivity of putrescine of the present invention was modified to enhance the activity of NCgl2522, which releases putrescine in cells, rather than endogenous activity, thereby increasing the release of putrescine into the cell, It was also confirmed that the resistance to.
- NCgl2522 in E. coli including the putrescine production pathway of the present invention it was confirmed that the amount of putrescine out of the cell increases. Therefore, NCgl2522 derived from Corynebacterium glutamicum can be widely used for the production of effective putrescine by applying to microorganisms having putrescine production ability.
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Abstract
Description
프라이머 | 서열(5'-3') |
NCgl1469-del-F1_BamHI(서열번호: 1) | CGGGATCCAACCTTCAGAACGCGAATAC |
NCgl1469-del-R1_SalI(서열번호: 2) | CGCGTCGACTTGGCACTGTGATTACCATC |
NCgl1469-del-F2_SalI(서열번호: 3) | CGCGTCGACTTGGGTTATATCCCCTCAGA |
NCgl1469-del-R2_XbaI(서열번호: 4) | TGCTCTAGATAGTGAGCCAAGACATGGAA |
프라이머 | 서열(5'-3') |
NCgl2522-del-F1_BamHI(서열번호: 5) | CGGGATCCCACGCCTGTCTGGTCGC |
NCgl2522-del-R1_SalI(서열번호: 6) | ACGCGTCGACGGATCGTAACTGTAACGAATGG |
NCgl2522-del-F2_SalI(서열번호: 7) | ACGCGTCGACCGCGTGCATCTTTGGACAC |
NCgl2522-del-R2_XbaI(서열번호: 8) | CTAGTCTAGAGAGCTGCACCAGGTAGACG |
Host | Genotype | Putrescine(g/L) |
KCCM11138P | (-) | 9.8 |
△NCgl2522 | 3.0 | |
KCCM11240P(KCCM11138P △NCgl1469) | (-) | 12.4 |
△NCgl2522 | 1.5 | |
DAB12-a | (-) | 10.2 |
△NCgl2522 | 0.7 | |
DAB12-b(DAB12-a △NCgl1469) | (-) | 13.1 |
△NCgl2522 | 0.3 |
프라이머 | 서열(5'-3') |
1'NCgl2522-F-T(서열번호: 9) | TGTCGGGCCCACTAGTGGTGCGACTTCAATTGTGCTCTT |
NCgl2522-R-T(서열번호: 10) | GAATGAGTTCCTCGAGCTAGTGCGCATTATTGGCTCC |
프라이머 | 서열(5'-3') |
NCgl2522-L5(서열번호: 11) | TGCAGGTCGACTCTAGAGTTCTGCGTAGCTGTGTGCC |
NCgl2522-L3(서열번호: 12) | GGATCGTAACTGTAACGAATGG |
CJ7-F(서열번호: 13) | CGTTACAGTTACGATCCAGAAACATCCCAGCGCTACTAATA |
CJ7-R(서열번호: 14) | AGTGTTTCCTTTCGTTGGGTACG |
NCgl2522-R5(서열번호: 15) | CAACGAAAGGAAACACTATGACTTCAGAAACCTTACAGGCG |
NCgl2522-R3(서열번호: 16) | TCGGTACCCGGGGATCCCACAAAAAGCGTAGCGATCAACG |
프라이머 | 서열(5'-3') |
2'NCgl2522-F-T(서열번호: 17) | TGTCGGGCCCACTAGTCTTCAATTCGAGTTGCTGCCAC |
NCgl2522-R-T(서열번호: 10) | GAATGAGTTCCTCGAGCTAGTGCGCATTATTGGCTCC |
프라이머 | 서열(5'-3') |
2'NCgl2522-L5(서열번호: 18) | TGCAGGTCGACTCTAGACAATTCGAGTTGCTGCCACAC |
NCgl2522-L3(서열번호: 12) | GGATCGTAACTGTAACGAATGG |
CJ7-F(서열번호: 13) | CGTTACAGTTACGATCCAGAAACATCCCAGCGCTACTAATA |
CJ7-R(서열번호: 14) | AGTGTTTCCTTTCGTTGGGTACG |
NCgl2522-R5(서열번호: 19) | CAACGAAAGGAAACACTATGATTTCAGAAACTTTGCAGGCG |
NCgl2522-R3(서열번호: 17) | TCGGTACCCGGGGATCCCACAAAAAGCGTAGCGATCAACG |
Host | Genotype | Putrescine(g/L) |
KCCM11138P | (-) | 9.8 |
Tn:1'NCgl2522 | 11.7 | |
P(CJ7)-NCgl2522 | 13.5 | |
KCCM11240P | (-) | 12.4 |
P(CJ7)-NCgl2522 | 15.5 | |
DAB12-a | (-) | 10.2 |
Tn:2'NCgl2522 | 12.3 | |
P(CJ7)-NCgl2522 | 14.1 | |
DAB12-b | (-) | 13.1 |
P(CJ7)-NCgl2522 | 15.9 |
Strain | Putrescine(mM) |
KCCM11138P | 7 |
KCCM11138P Tn:1'NCgl2522 | 2 |
프라이머 | 서열(5'-3') |
SPEC-F(서열번호: 34) | CACAGGAAACAGACCATGGATGAAATCAATGAATATTGCCGCCA |
SPEC-R(서열번호: 35) | GTGCAGGTGCTGAATTCTTACTTCAACACATAACCGTACAAC |
Ptrc-F(서열번호: 36) | TGCAGGCATGCAAGCTTCGACATCATAACGGTTCTGGC |
Ptrc-R(서열번호: 37) | ATTATACGAGCCGGATGATTAATTG |
NCgl2522-F(서열번호: 38) | CATCCGGCTCGTATAATATGACTTCAGAAACCTTACAGGC |
NCgl2522-R(서열번호: 39) | ATAGAATACTCAAGCTTCTAGTGCGCATTATTGGCTCC |
Host | Plasmid | Putrescine(mg/L) |
W3110 | (-) | 11 |
pSE280-speC | 56 | |
pcc1BAC-P(trc)-NCgl2522 | 250 |
Claims (10)
- 서열번호: 21 또는 23으로 기재되는 아미노산 서열을 포함하는 단백질의 활성이 강화되도록 변형된, 퓨트레신 생산능을 갖는 미생물.
- 제1항에 있어서, 상기 미생물이 추가로 오르니틴 카르바모일 트렌스퍼라아제(ArgF) 및 글루타메이트 배출에 관여하는 단백질(NCgl1221)의 활성이 내재적 활성에 비해 약화되도록 변형되고, 오르니틴 디카복실라아제(ODC)의 활성이 강화된 것인, 퓨트레신 생산능을 갖는 미생물.
- 제2항에 있어서, 상기 오르니틴 카르바모일 트렌스퍼라아제(ArgF)가 서열번호: 29로 기재되는 아미노산 서열을 포함하고, 글루타메이트 배출에 관여하는 단백질(NCgl1221)이 서열번호: 30으로 기재되는 아미노산 서열을 포함하고, 오르니틴 디카복실라아제(ODC)가 서열번호: 33으로 기재되는 아미노산 서열을 포함하는 것인, 퓨트레신 생산능을 갖는 미생물.
- 제1항에 있어서, 상기 미생물이 추가적으로 아세틸 감마 글루타밀 포스페이트 리덕타아제(ArgC), 아세틸글루타메이트 신타아제 또는 오르니틴 아세틸트랜스퍼라아제(ArgJ), 아세틸글루타메이트 키나아제(ArgB), 및 아세틸오르니틴 아미노트랜스퍼라아제(ArgD)의 활성이 내재적 활성에 비해 강화되도록 변형된 것인, 퓨트레신 생산능을 갖는 미생물.
- 제4항에 있어서, 상기 아세틸 감마 글루타밀 포스페이트 리덕타아제(ArgC), 아세틸글루타메이트 신타아제 또는 오르니틴 아세틸트랜스퍼라아제(ArgJ), 아세틸글루타메이트 키나아제(ArgB), 및 아세틸오르니틴 아미노트랜스퍼라아제(ArgD)가 각각 서열번호: 25, 26, 27 및 28로 기재되는 아미노산 서열을 포함하는 것인, 퓨트레신 생산능을 갖는 미생물.
- 제1항에 있어서, 상기 미생물이 추가적으로 아세틸트렌스퍼라아제(NCgl1469)의 활성이 약화된 것인, 퓨트레신 생산능을 갖는 미생물.
- 제6항에 있어서, 상기 아세틸트렌스퍼라아제(NCgl1469)가 서열번호: 31 또는 32로 기재되는 아미노산 서열을 포함하는 것인, 퓨트레신 생산능을 갖는 미생물.
- 제1항에 있어서, 상기 미생물이 에스케리키아 속 미생물 또는 코리네형 미생물인 것인, 퓨트레신 생산능을 갖는 미생물.
- 제8항에 있어서, 상기 미생물이 대장균 또는 코리네박테리움 글루타미쿰인 것인, 퓨트레신 생산능을 갖는 미생물.
- (i) 제1항 내지 제9항 중 어느 한 항에 따른 퓨트레신 생산능을 갖는 미생물을 배양하여 배양물을 수득하는 단계; 및(ii) 상기 배양된 미생물 또는 배양물로부터 퓨트레신을 회수하는 단계를 포함하는, 퓨트레신의 생산방법.
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Also Published As
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TW201441367A (zh) | 2014-11-01 |
US10221433B2 (en) | 2019-03-05 |
CN105492593B (zh) | 2019-08-09 |
BR112015018599A2 (pt) | 2017-08-22 |
RU2665825C2 (ru) | 2018-09-04 |
US20170002386A1 (en) | 2017-01-05 |
EP2977443B1 (en) | 2020-04-08 |
CN105492593A (zh) | 2016-04-13 |
KR101607741B1 (ko) | 2016-03-31 |
AU2014238707A1 (en) | 2015-07-23 |
HUE048852T2 (hu) | 2020-08-28 |
EP2977443A4 (en) | 2016-09-07 |
TWI657142B (zh) | 2019-04-21 |
AU2014238707B2 (en) | 2017-08-31 |
MY172080A (en) | 2019-11-13 |
TW201623617A (zh) | 2016-07-01 |
EP2977443A1 (en) | 2016-01-27 |
JP2016514466A (ja) | 2016-05-23 |
RU2015142261A (ru) | 2017-04-26 |
US11053524B2 (en) | 2021-07-06 |
JP6297134B2 (ja) | 2018-03-20 |
KR20140115244A (ko) | 2014-09-30 |
US20190136274A1 (en) | 2019-05-09 |
BR112015018599B1 (pt) | 2022-12-13 |
TWI632238B (zh) | 2018-08-11 |
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