Patent Grant
11053524
The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is HANO_035_00US_ST25.txt. The text file is 48 KB, created on Jan. 11, 2019, and is being submitted electronically via EFS-Web.
The present invention relates to a recombinant microorganism having improved putrescine productivity and a method for producing putrescine at a high yield using the same.
Polyamines such as spermidine, spermine or the like are present in most living cells, and putrescine (or 1,4-butanediamine) is used as a precursor in spermidine and spermine metabolisms. Putrescine is found in Gram-negative bacteria or fungus, and it is present in high concentrations in various species, suggesting that it has an important role in the metabolic pathways of microorganisms.
In general, putrescine is an important raw material in a synthesis of polyamine nylon-4, 6 which is produced by reacting with adipic acid. Putrescine is produced mainly by chemical synthesis through acrylonitrile and succinonitrile from propylene. This chemical synthesis is a three-step process including a catalytic oxidation reaction, a reaction using a cyanide compound, and a hydrogenation reaction using high-pressure hydrogen. There are problems in that this chemical synthesis is not environment friendly and also consumes a lot of energy leading to depletion of petroleum. Therefore, a more environment friendly and energy-effective method involving biomass utilization needs to be developed for putrescine production.
In microorganisms, a biosynthetic pathway of putrescine is the same as route of arginine synthesis from glutamate to ornithine synthesis. Putrescine can be biosynthesized through two pathways from microorganisms. In one pathway, ornithine as an intermediate is decarboxylated to synthesize putrescine. In the other pathway, agmatine is produced by decarboxylation arginine synthesized from ornithine, and then putrescine is synthesized from the agmatine (Morris et al., J Biol. Chem. 241: 13, 3129-3135, 1996). These two pathways produce the energy required for metabolism or allow the cell to have resistance to oxidative stress.
As a method for producing putrescine using a microorganism, a method for producing putrescine at a high concentration by transformation of E. coli and Corynebacterium has been reported (International Patent Publication No. WO06/005603; International Patent Publication No. WO09/125924; Qian Z D et al., Biotechnol. Bioeng. 104: 4, 651-662, 2009; Schneider et al., Appl. Microbiol. Biotechnol. 88: 4, 859-868, 2010; Schneider et al., Appl. Microbiol. Biotechnol. 91: 17-30, 2011). For example, WO09/125924 discloses a method for producing putrescine in a high yield by enhancing ornithine biosynthetic pathway, instead of inactivating pathways involved in degradation and utilization of putrescine which are present in E. coli and inactivating conversion of ornithine as a precursor of putrescine to arginine. In addition, Schneider (2010) discloses a method for producing putrescine at a high concentration by introducing and enhancing a protein capable of converting ornithine to putrescine into a Corynebacterium sp. strain having no putrescine productivity.
Furthermore, studies on putrescine transporters in E. coli, yeast, plant and animal cells have been actively conducted (K Igarashi, Plant Physiol. Biochem. 48: 506-512, 2010). Putrescine uptake of E. coli occurs via 4 pathways; potABCD or potFGHI driven by ATP hydrolysis, andpotE as H+ symporter and puuP of the puu pathway. With regard to Km values of these complexes involved in putrescine uptake, those of PotFGHI, potABCD, potE and puuP are 0.5 mM, 1.5 mM, 1.8 mM, and 3.7 mM, respectively. Among the four putrescine uptake pathways, potFGHI complex is considered as the most suitable. In addition, potE transporter has both functions of uptake and excretion of putrescine. Putrescine is imported together with proton into cells at neural pH. However, as putrescine synthase (speF) is expressed under acidic pH conditions, intracellular uptake of extracellular ornithine and extracellular excretion of putrescine synthesized within cells occur at the same time (Kurihara et. al., J. Bacteriology 191: 8, 2776-2782, 2009).
The known putrescine exporters in yeast are TPO1 and TPO4. These amino acid sequence are very similar to the amino acid sequence of bacillus multidrug transporter Blt.
These two exporters share characteristics with potE in E. coli, and they have functions of importing putrescine, spermidine, and spermine under basic conditions and exporting them under acidic conditions. In addition, yeast cell over-expressing TPO5 gene is resistant to 120 mM putrescine whereas a mutant disrupted TPO5 gene is sensitive to 90 mM putrescine (Tachihara et. al., J. Biological Chemistry, 280(13): 12637-12642, 2005).
Synthesis and degradation, and uptake and excretion of putrescine in animal cells are regulated in various ways. Although studies on polyamine excretion have not been done in animal cells as well as in E. coli or yeast, there is a report that an SLC3A2 (arginine/diamine exporter) functions to import arginine into cells and to export putrescine, acetyl spermidine, and acetyl spermine in colon epithelial cells. However, there has been no report about uptake and export of putrescine in plant cells (Igarashi et al., Plant Physiol. & Biochem. 48: 506-512, 2010).
On the other hand, since Corynebacterium sp. microorganism has no putrescine biosynthetic pathway, studies regarding putrescine export have not been studied. According to a recent report, cell growth is restored and cadaverine productivity is increased by overexpression of a cg2983 membrane protein in a strain producing a cadaverine (Kind et. al., Metabolic Engineering 13: 617-627, 2011).
However, there have been no reports about association between putrescine exporter and putrescine productivity or growth of microorganisms producing putrescine. In the above literature, there is no mention about association between cg2983 membrane protein and the exporting ability of putrescine.
In this background, the present inventors have made many efforts to develop a strain capable of producing putrescine in a higher yield. As a result, NCgl2522 functions is revealed as a putrescine exporter in a putrescine-producing strain, Corynebacterium sp. microorganism, and putrescine can be produced in a high yield by enhancing NCgl2522 activity, compared to the endogenous activity thereof. In addition, the amount of putrescine in a culture medium can be increased by expressing NCgl2522 in E. coli having the putrescine synthetic pathway, and thus the present inventors suggested that NCgl2522 also functions as a putrescine exporter in E. coli, thereby completing the present invention.
An object of the present invention is to provide a recombinant microorganism which is modified to have enhanced NCgl2522 activity, thereby produced putrescine in a high yield. Another object of the present invention is to provide a method for producing putrescine in a high yield using the microorganism.
1: KCCM11240P
2: KCCM11240P ΔNCgl2522
3: KCCM11240P P(CJ7)-NCgl2522
In one aspect to achieve the above object, the present invention provides a microorganism having putrescine productivity, which is modified to enhance activity of a protein having an amino acid sequence represented by SEQ ID NO: 21 or 23.
In one specific embodiment, the present invention provides a microorganism having putrescine productivity, in which the microorganism is further modified to have weakened activities of ornithine carbamoyltransferase (ArgF) and a protein (NCgl1221) involved in glutamate export, compared to the endogenous activities thereof, and is introduced with ornithine decarboxylase (ODC) activity.
In another specific embodiment, the present invention provides a microorganism having putrescine productivity, in which the ornithine carbamoyltransferase (ArgF) has an amino acid sequence represented by SEQ ID NO: 29, the protein (NCgl1221) involved in glutamate export has an amino acid sequence represented by SEQ ID NO: 30, and the ornithine decarboxylase (ODC) has an amino acid sequence represented by SEQ ID NO: 33.
In still another specific embodiment, the present invention provides a microorganism having putrescine productivity, in which the microorganism is further modified to have enhanced activities of acetyl-gamma-glutamyl-phosphate reductase (ArgC), acetylglutamate synthase or ornithine acetyltransferase (ArgJ), acetylglutamate kinase (ArgB), and acetylornithine aminotransferase (ArgD), compared to the endogenous activities thereof.
In still another specific embodiment, the present invention provides a microorganism having putrescine productivity, in which the acetyl-gamma-glutamyl-phosphate reductase (ArgC), acetylglutamate synthase or ornithine acetyltransferase (ArgJ), acetylglutamate kinase (ArgB), and acetylornithine aminotransferase (ArgD) have amino acid sequences represented by SEQ ID NOs: 25, 26, 27 and 28, respectively.
In still another specific embodiment, the present invention provides a microorganism having putrescine productivity, in which acetyltransferase (NCgl1469) activity of the microorganism is further weakened.
In still another specific embodiment, the present invention provides a microorganism having putrescine productivity, in which the acetyltransferase has an amino acid sequence represented by SEQ ID NO: 31 or 32.
In still another specific embodiment, the present invention provides a microorganism having putrescine productivity, in which the microorganism is an Escherichia sp. or a Corynebacterium sp.
In still another specific embodiment, the present invention provides a microorganism having putrescine productivity, in which the microorganism is E. coli or Corynebacterium glutamicum.
In another aspect, the present invention provides a method for producing putrescine, comprising the steps of culturing a microorganism having putrescine productivity to obtain a cell culture and recovering putrescine from the cultured microorganism or cell culture.
Hereinafter, the present invention will be described in detail.
The present invention provides a recombinant Corynebacterium sp. microorganism, in which the Corynebacterium sp. microorganism having putrescine productivity is modified to have enhanced NCgl2522 activity, compared to the endogenous activity thereof and thus it has improved putrescine productivity.
As used herein, the term “NCgl2522” refers to permease belonging to MFS (major facilitator superfamily), which is a membrane protein isolated from Corynebacterium glutamicum ATCC13032. NCgl2522 is known to export diaminopentane from Corynebacterium glutamicum. In the present invention, NCgl2522 was confirmed to function as a transporter that serves to extracellularly export putrescine produced within cells. On the basis of this fact, the present invention provides a recombinant microorganism showing high-yield putrescine productivity, in which NCgl2522 is modified to have enhanced activity, compared to the endogenous activity thereof, and therefore, export of intracellularly produced putrescine is increased.
As used herein, the term “endogenous activity” refers to the activity of an enzyme that a microorganism possesses in its native state, namely in the state without modification, and the meaning of “modified to have enhanced activity, compared to the endogenous activity” is that the activity of the enzyme is newly introduced or further improved, compared to the activity of the corresponding enzyme before modification.
In the present invention, “enhancement of enzymatic activity” includes improvement in the enzymatic activity by improvement in endogenous gene activity, amplification of the endogenous gene by internal or external factors, deletion of a regulatory factor for suppressing the gene expression, increase in the gene copy number, increase in the activity by introduction of a foreign gene or modification of an expression regulatory sequence, in particular, replacement or modification of a promoter and mutation within gene, as well as introduction or improvement of the activity of the enzyme itself to achieve effects beyond the endogenous functions.
In the present invention, “modified to have enhanced activity, compared to the endogenous activity” means that the activity of the microorganism is increased after manipulation such as introduction of a gene showing the activity, or increase in the gene copy number, deletion of a regulatory factor for suppressing the gene expression or modification of an expression regulatory sequence, for example, use of an improved promoter, compared to the activity of the microorganism before the manipulation.
The NCgl2522, having its activity is increased by the present invention, may be, but is not particularly limited to, a protein having an amino acid sequence of SEQ ID NO: 21 or 23 or an amino acid sequence having 70% or more homology thereto, preferably 80% or more homology thereto, more preferably 90% or more homology thereto, much more preferably 95% or more homology thereto, much more preferably 98% or more homology thereto, and most preferably 99% or more homology thereto. Further, because the amino acid sequence of the protein showing the activity may differ depending on species or strain of the microorganism, the protein is not limited thereto. That is, the protein may be a protein mutant or an artificial variant that has an amino acid sequence including substitution, deletion, insertion, or addition of one or several amino acids at one or more positions of the amino acid sequence of SEQ ID NO: 21 or 23, as long as the protein aids to improve putrescine productivity by enhancing its activity. As used herein, the term “several” amino acids means specifically 2 to 20, preferably 2 to 10, and more preferably 2 to 5 amino acids, although it may differ depending on the position or type of amino acid residue in the three-dimensional structure of the protein. Furthermore, the substitution, deletion, insertion, addition or inversion of amino acids may include naturally occurring mutations which occur due to differences of individual or species of the microorganism having the activity of the polypeptide or artificial variation.
There are no putrescine biosynthetic pathways in Corynebacterium sp. microorganism. However, when external ornithine decarboxylase (ODC) is introduced, putrescine is synthesized and excreted extracellularly, indicating presence of a transporter, that is, an exporter that functions as a passage of putrescine among numerous membrane proteins of Corynebacterium sp. microorganism. Accordingly, in order to isolate the putrescine exporter in Corynebacterium sp. microorganism, the present inventors prepared a chromosome library of the wild-type Corynebacterium glutamicum ATCC13032, and they transformed a putrescine-producing strain, Corynebacterium glutamicum KCCM11138P with the library, and selected strains that grow in a minimal medium containing putrescine. Through tertiary colony selection, a clone (B19) having putrescine resistance was finally selected and base sequence analysis was performed to confirm that the clone contains NCgl2522 (see
A polynucleotide encoding NCgl2522 of the present invention may include a polynucleotide encoding the protein having the amino acid sequence of SEQ ID NO: 21 or 23, or the amino acid sequence having 70% or more homology thereto, preferably 80% or more homology thereto, more preferably 90% or more homology thereto, much more preferably 95% or more homology thereto, much more preferably 98% or more homology thereto, and most preferably 99% or more homology thereto, as long as the protein has the activity similar to that of the NCgl2522 protein, and most preferably, it may include a nucleotide sequence of SEQ ID NO: 20 or 22.
As used herein, the term “homology” refers to the similarity between two amino acid sequences, and can be determined using the well-known methods using BLAST 2.0, which calculates parameters such as score, identity, and similarity.
Further, the polynucleotide encoding NCgl2522 of the present invention may be a variant which hybridizes under stringent conditions with the nucleotide sequence of SEQ ID NO: 20 or 22, or a probe derived from the above nucleotide sequence, provided that it encodes a functional NCgl2522. As used herein, the term “stringent conditions” mean conditions allowing a specific hybridization between polynucleotides. For example, such stringent conditions are described in detail in the literature (J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, N.Y., 1989; F. M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).
In the present invention, “modified to have enhanced NCgl2522 activity, compared to the endogenous activity” may be performed by a method selected from methods of increasing the copy number of the polynucleotide encoding the protein, modifying an expression regulatory sequence to increase expression of the polynucleotide, modifying the polynucleotide sequence on the chromosome to enhance the activity of the enzyme, deleting a regulatory factor for suppressing the gene expression, and combinations thereof.
The copy number of the polynucleotide may be, but is not particularly limited to, increased by operably linking the polynucleotide to a vector or by integrating it into the host cell genome. Specifically, the copy number of the polynucleotide in the host cell genome can be increased by introducing into the host cell the vector which is operably linked to the polynucleotide encoding the protein of the present invention and replicates and functions independently of the host cell, or by introducing into the host cell the vector which is operably linked to the polynucleotide and is able to integrate the polynucleotide into the host cell genome.
As used herein, the term “vector” refers to a DNA construct including a nucleotide sequence encoding the desired protein, which is operably linked to an appropriate expression regulatory sequence to express the desired protein in a suitable host cell. The regulatory sequence includes a promoter that can initiate transcription, an optional operator sequence for regulating the transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. After the vector is transformed into the suitable host cell, it can replicate or function independently of the host genome, and can be integrated into the genome itself.
The vector used in the present invention is not particularly limited, as long as it is able to replicate in the host cell, and any vector known in the art can be used. Examples of conventional vectors may include a natural or recombinant plasmid, cosmid, virus and bacteriophage. For instance, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as a phage vector or cosmid vector. As a plasmid vector, pBR type, pUC type, pBluescriptII type, pGEM type, pTZ type, pCL type and pET type may be used. A vector usable in the present invention is not particularly limited, and any known expression vector can be used. Preferably, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, or pCC1BAC vector may be used, and more preferably, pDZ vector may be used.
Further, the polynucleotide encoding the desired protein in the chromosome can be replaced by a mutated polynucleotide using a vector for chromosomal insertion. The insertion of the polynucleotide into the chromosome may be performed by any method known in the art, for example, homologous recombination. Since the vector of the present invention can be inserted into the chromosome by homologous recombination, it may further include a selection marker to confirm chromosomal insertion. The selection marker is to select cells transformed with the vector, that is, to confirm insertion of the desired polynucleotide, and the selection marker may include markers providing selectable phenotypes, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or surface protein expression. Only cells expressing the selection marker are able to survive or to show different phenotypes under the environment treated with the selective agent, and thus the transformed cells can be selected.
As used herein, the term “transformation” means the introduction of a vector including a polynucleotide encoding a target protein into a host cell in such a way that the protein encoded by the polynucleotide is expressed in the host cell. As long as the transformed polynucleotide can be expressed in the host cell, it can be either integrated into or placed in the chromosome of the host cell, or exist extrachromosomally. Further, the polynucleotide includes DNA and RNA encoding the target protein. The polynucleotide can be introduced in any form, as long as it can be introduced into the host cell and expressed therein. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a gene construct including all elements required for its autonomous expression. Typically, the expression cassette includes a promoter operably linked to the polynucleotide, transcriptional termination signals, ribosome binding sites, or translation termination signals. The expression cassette may be in the form of a self-replicable expression vector. Also, the polynucleotide as it is may be introduced into the host cell and operably linked to sequences required for expression in the host cell.
Further, as used herein, the term “operably linked” means a functional linkage between a polynucleotide sequence encoding the desired protein and a promoter sequence which initiates and mediates transcription of the polynucleotide sequence.
As well, modification of the expression regulatory sequence for increasing the polynucleotide expression may be, but is not limited to, done by inducing a modification on the expression regulatory sequence through deletion, insertion, non-conservative or conservative substitution of nucleotide sequence, or a combination thereof in order to further enhance the activity of expression regulatory sequence, or by replacing the expression regulatory sequence with a nucleotide sequence having stronger activity. The expression regulatory sequence includes, but is not particularly limited to, a promoter, an operator sequence, a sequence coding for ribosome-binding site, and a sequence regulating the termination of transcription and translation.
A strong heterologous promoter instead of the original promoter may be linked upstream of the polynucleotide expression unit, and examples of the strong promoter may include CJ7 promoter, lysCP1 promoter, EF-Tu promoter, groEL promoter, aceA or aceB promoter, and more preferably, lysCP1 promoter or CJ7 promoter as a Corynebacterium-derived promoter, and the polynucleotide encoding the enzyme is operably linked thereto so that its expression rate can be increased. Herein, the lysCP1 promoter is a promoter improved through nucleotide sequence substitution of the promoter region of the polynucleotide encoding aspartate kinase and aspartate semialdehyde dehydrogenase, and is a strong promoter that increases expression of the aspartate kinase gene, leading to 5-fold increased activity of the corresponding enzyme, compared to the wild-type (WO 2009/096689). Further, CJ7 promoter is a promoter that was found during exploration of a strong promoter sequence in Corynebacterium ammoniagenes and confirmed to be expressed in Corynebacterium ammoniagenes and Escherichia and to have a strong promoter activity. CJ7 promoter is a promoter that also shows high expression activity in Corynebacterium glutamicum (Korean Patent No. 0620092 and WO 2006/065095).
Furthermore, modification of a polynucleotide sequence on chromosome may be, but is not particularly limited to, done by inducing a mutation on the expression regulatory sequence through deletion, insertion, non-conservative or conservative substitution of polynucleotide sequence, or a combination thereof in order to further enhance the activity of the polynucleotide sequence, or by replacing the sequence with a polynucleotide sequence which is modified to have stronger activity.
In one preferred embodiment of the present invention, in order to provide a Corynebacterium sp. microorganism having improved putrescine productivity, the copy number of the gene can be increased by introducing into the chromosome the polynucleotide having the nucleotide sequence of SEQ ID NO: 20 or 22 encoding NCgl2522 involved in putrescine excretion, or the own promoter of NCgl2522 can be substituted with a promoter having improved activity, preferably, CJ7 promoter having the nucleotide sequence of SEQ ID NO: 24.
As used herein, the term “microorganism having putrescine productivity” or “microorganism producing putrescine” refers to a microorganism that is prepared by providing putrescine productivity for the parent strain having no putrescine productivity. The microorganism that is provided with putrescine productivity or produces putrescine may be, but is not particularly limited to, a microorganism having improved productivity of ornithine to be used as a raw material for putrescine biosynthesis, in which the microorganism is modified to have higher activities of acetylglutamate synthase converting glutamate to acetylglutamate (N-acetylglutamate) or omithine acetyltransferase (ArgJ) converting acetyl ornithine to ornithine, acetylglutamate kinase (ArgB) converting acetyl glutamate to acetylglutamyl phosphate (N-acetylglutamyl phosphate), acetyl-gamma-glutamyl phosphate reductase (ArgC) converting acetyl glutamyl phosphate to acetyl glutamate semialdehyde (N-acetyl glutamate semialdehyde), or acetylomithine aminotransferase (ArgD) converting acetyl glutamate semialdehyde to acetylomithine (N-acetylomithine) than the endogenous activity, in order to enhance the biosynthetic pathway from glutamate to omithine. Further, the microorganism is a microorganism that is modified to have weaker activity of omithine carbamoyltransferase (ArgF) involved in synthesis of arginine from ornithine, the protein (NCgl1221) involved in glutamate excretion, and/or the protein (NCgl469) acetylating putrescine than the endogenous activity, and/or modified to have ornithine decarboxylase (ODC) activity.
In this regard, acetyl-gamma-glutamyl-phosphate reductase (ArgC), acetylglutamate synthase or ornithineacetyltransferase (ArgJ), acetylglutamate kinase (ArgB), acetylornithine aminotransferase (ArgD), ornithine carbamoyl transferase (ArgF), the protein (NCgl1221) involved in glutamate export, and ornithine decarboxylase (ODC) may have, but are not particularly limited to, preferably the amino acid sequences represented by SEQ ID NOs: 25, 26, 27, 28, 29, 30 and 33, respectively, or amino acid sequences having 70% or more homology thereto, more preferably 80% or more homology thereto, or much more preferably 90% or more homology thereto, respectively. In addition, the protein (NCgl469) acetylating putrescine may have, but is not particularly limited to, preferably the amino acid sequence represented by SEQ ID NO: 31 or 32, or an amino acid sequence having 70% or more homology thereto, more preferably 80% or more homology thereto, or much more preferably 90% or more homology thereto.
Of the proteins, the increase in the activities of acetyl-gamma-glutamyl-phosphate reductase (ArgC), acetylglutamate synthase or ornithineacetyltransferase (ArgJ), acetylglutamate kinase (ArgB), acetylornithine aminotransferase (ArgD), and ornithine decarboxylase (ODC) may be achieved by the above described method of increasing the NCgl2522 activity, for example, a method selected from the methods of increasing the copy number of the polynucleotide encoding the protein, modifying an expression regulatory sequence to increase expression of the polynucleotide, modifying the polynucleotide sequence on the chromosome to enhance the activity of the enzyme, deleting a regulatory factor to suppress the expression of the polynucleotide of the enzyme, and combinations thereof.
Further, activities of ornithine carbamoyl transferase (ArgF), the protein (NCgl1221) involved in glutamate export, and the protein (NCgl469) acetylating putrescine can be diminished by a method selected from the group consisting of a partial or full deletion of a polynucleotide encoding the protein, modification of an expression regulatory sequence for suppressing the polynucleotide expression, modification of the polynucleotide sequence on chromosome for diminishing the protein activity, and a combination thereof
In detail, a partial or full deletion of the polynucleotide encoding the protein can be done by introducing a vector for chromosomal insertion into a microorganism, thereby substituting the polynucleotide encoding an endogenous target protein on chromosome with a partially removed polynucleotide or a marker gene. The “partial” may vary depending on the type of polynucleotide, but specifically refers to 1 to 300, preferably 1 to 100, and more preferably 1 to 50 nucleotides.
Also, modification of the expression regulatory sequence can be done by inducing a modification on the expression regulatory sequence through deletion, insertion, non-conservative or conservative substitution of nucleotide sequence, or a combination thereof in order to diminish the activity of expression regulatory sequence, or by replacing the expression regulatory sequence with a nucleotide sequence having weaker activity. The expression regulatory sequence includes a promoter, an operator sequence, a sequence coding for ribosome-binding site, and a sequence regulating the termination of transcription and translation.
Furthermore, modification of a polynucleotide sequence on chromosome can be done by inducing a mutation on the sequence through deletion, insertion, non-conservative or conservative substitution of polynucleotide sequence, or a combination thereof in order to further diminish the enzymatic activity, or by replacing the sequence with a polynucleotide sequence which is modified to have weaker activity.
Moreover, a regulatory factor for suppressing the expression of the polynucleotide of the enzyme can be deleted by substituting a polynucleotide of the expression suppressing factor with a partially removed polynucleotide or a marker gene. The “partial” may vary depending on the type of polynucleotide, but specifically refers to 1 to 300, preferably 1 to 100, and more preferably 1 to 50 nucleotides.
Meanwhile, the microorganism of the present invention is a microorganism having putrescine productivity, and includes a prokaryotic microorganism expressing the protein having the amino acid sequence represented by SEQ ID NO: 21 or 23, and examples thereof may include microorganisms belonging to Escherichia sp., Shigella sp., Citrobacter sp., Salmonella sp., Enterobacter sp., Yersinia sp., Klebsiella sp., Erwinia sp., Corynebacterium sp., Brevibacterium sp., Lactobacillus sp., Selenomanas sp., Vibrio sp., Pseudomonas sp., Streptomyces sp., Arcanobacterium sp., Alcaligenes sp. or the like. The microorganism of the present invention is preferably a microorganism belonging to Escherichia sp. or a microorganism belonging to Corynebacterium sp., and more preferably, E. coli or Corynebacterium glutamicum.
In a specific embodiment of the present invention, a Corynebacterium sp. microorganism with Accession No. KCCM11138P (Korean Patent Publication NO. 2012-0064046) and a Corynebacterium sp. microorganism with Accession No. KCCM11240P (Korean Patent Application NO. 2012-0003634) were used as strains that have enhanced synthetic pathway from glutamate to putrescine, thereby producing putrescine at a high concentration.
In still another embodiment of the present invention, Corynebacterium glutamicum ATCC13032-based putrescine-producing strains, KCCM11138P and KCCM11240P, and Corynebacterium glutamicum ATCC13869-based putrescine-producing strains DAB12-a and DAB12-b having the same genotype were used. ATCC13869 strain can be obtained from American Type Culture Collection (ATCC). That is, a unique accession number is listed in the catalog of ATCC is given for each strain, and the strain can be ordered using the accession number. Specifically, the putrescine-producing strain DAB12-a is characterized by deletion of a gene encoding ornithine carbamoyl transferase (ArgF) and a gene encoding the glutamate exporter NCgl1221, introduction of a gene encoding ornithine decarboxylase (OCD), and replacement of the promoter of ornithine biosynthetic gene operon (argCJBD) by an improved promoter in the Corynebacterium glutamicum ATCC13869. Further, the putrescine-producing strain DAB12-b is characterized in that it is prepared by modifying the DAB12-a strain to have weakened activity of the protein (NCgl1469) acetylating putrescine, compared to the endogenous activity.
According to one preferred Example, Corynebacterium glutamicum KCCM11138P prepared by deletion of the gene encoding ornithine carbamoyl transferase (ArgF) and a gene encoding the glutamate exporter NCgl1221, replacement of the own promoter of ArgCJBD gene cluster encoding an enzyme involved in the synthesis of ornithine from glutamate by an improved promoter, and introduction of the gene encoding ornithine decarboxylase (ODC) into the chromosome in the wild-type Corynebacterium glutamicum ATCC13032, and Corynebacterium glutamicum KCCM11240P prepared by additionally weakening a gene encoding the acetyltransferase NCgl1469 in the microorganism were prepared as putrescine-producing strains.
Meanwhile, in order to prepare an NCgl2522—deleted strain derived from Corynebacterium glutamicum ATCC13032, a plasmid pDZ-1′NCgl2522(K/O) was prepared, based on the nucleotide sequence of NCgl2522 derived from Corynebacterium glutamicum ATCC13032.
The plasmid pDZ-1′NCgl2522(K/O) was transformed into the prepared putrescine-producing strains, KCCM11138P and KCCM11240P, and selected as NCgl2522—deleted strains, and these strains were designated as KCCM11138P ΔNCgl2522 and KCCM11240P ΔNCgl2522, respectively. In the same manner, NCgl2522—deleted strains derived from Corynebacterium glutamicum ATCC13869 were prepared and designated as DAB12-a ΔNCgl2522 and DAB12-b ΔNCgl2522.
Putrescine productivities of 4 types of NCgl2522—deleted strains thus prepared were compared with that of the parent strain, and as a result, putrescine productivity was reduced in all of NCgl2522—deleted KCCM11138P ΔNCgl2522, KCCM11240P ΔNCgl2522, DAB12-a ΔNCgl2522, and DAB12-b ΔNCgl2522, compared to the parent strain (see Table 3). Based on this result, the present inventors confirmed that NCgl2522 activity in the putrescine-producing strain is closely related to putrescine productivity, and they prepared NCgl2522-enhanced strains in order to increase putrescine productivity through enhancement of the activity.
To this end, in one preferred Example of the present invention, NCgl2522 was additionally introduced into the transposon of Corynebacterium glutamicum strain or the own NCgl2522 promoter within the chromosome was replaced by the CJ7 promoter (KCCM10617, Korean Patent NO. 10-0620092) that was newly developed by the present inventors.
Putrescine productivities of 6 types of NCgl2522-enhanced strains thus prepared were compared with that of the parent strain, and as a result, putrescine productivity was increased in all of the strains prepared by additional introduction of NCgl2522 into the transposon, compared to the parent strain (see Table 6). Intracellular putrescine concentrations were measured in the NCgl2522-enhanced strains showing an improvement in putrescine productivity, and as a result, they showed reductions of intracellular putrescine concentrations, compared to the parent strain (see Table 9). Based on these results, the present inventors confirmed that extracellular export of putrescine intracellularly produced is increased by enhancing NCgl2522 activity in the putrescine-producing strains, thereby improving putrescine productivity.
Accordingly, the Corynebacterium sp. microorganism having an enhanced putrescine productivity, in which the putrescine-producing strain Corynebacterium glutamicum KCCM11138P was modified to have enhanced NCgl2522 activity, compared to the endogenous activity, and thus exhibits an enhanced ability to export putrescine, was designated as Corynebacterium glutamicum CC01-0510, and deposited under the Budapest Treaty to the Korean Culture Center of Microorganisms (KCCM) on Mar. 8, 2013, with Accession No. KCCM11401P.
According to another aspect of the present invention, the present invention provides a method for producing putrescine, including the steps of:
(i) culturing the microorganism having putrescine productivity to obtain a cell culture; and
(ii) recovering putrescine from the cultured microorganism or the cell culture.
In the method, the step of culturing the microorganism may be, but is not particularly limited to, preferably performed by batch culture, continuous culture, and fed-batch culture known in the art. In this regard, the culture conditions are not particularly limited, but an optimal pH (e.g., pH 5 to 9, preferably pH 6 to 8, and most preferably pH 6.8) can be maintained by using a basic chemical (e.g., sodium hydroxide, potassium hydroxide or ammonia) or acidic chemical (e.g., phosphoric acid or sulfuric acid). Also, an aerobic condition can be maintained by adding oxygen or oxygen-containing gas mixture to a cell culture. The culture temperature may be maintained at 20 to 45° C., and preferably at 25 to 40° C. In addition, the cultivation is preferably performed for about 10 to 160 hours. The putrescine produced by the above cultivation may be excreted to a culture medium or remain inside the cell.
Furthermore, the culture medium to be used may include sugar and carbohydrate (e.g., glucose, sucrose, lactose, fructose, maltose, molasse, starch and cellulose), oil and fat (e.g., soybean oil, sunflower seed oil, peanut oil and coconut oil), fatty acid (e.g., palmitic acid, stearic acid and linoleic acid), alcohol (e.g., glycerol and ethanol), and organic acid (e.g., acetic acid) individually or in combination as a carbon source; nitrogen-containing organic compound (e.g., peptone, yeast extract, meat juice, malt extract, corn solution, soybean meal powder and urea), or inorganic compound (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate) individually or in combination as a nitrogen source; potassium dihydrogen phosphate, dipotassium phosphate, or sodium-containing salt corresponding thereto individually or in combination as a phosphorus source; other essential growth-stimulating substances including metal salts (e.g., magnesium sulfate or iron sulfate), amino acids, and vitamins.
The method for recovering putrescine that is produced in the culturing step of the present invention can be carried out, for example, using a suitable method known in the art according to batch culture, continuous culture, or fed-batch culture, thereby collecting the desired amino acids from the culture.
Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are for illustrative purposes only, and the invention is not intended to be limited by these Examples.
In order to prepare a Corynebacterium sp. microorganism having putrescine productivity, the biosynthetic pathway of arginine from ornithine was blocked, the biosynthetic pathway of ornithine from glutamate was enhanced, and the foreign ornithine decarboxylase (OCD) was introduced to prepare a microorganism provided with putrescine productivity, as described in Korean Patent Publication NO. 10-2012-0064046.
Specifically, based on the Corynebacterium glutamicum ATCC13032, a gene encoding ornithine carbamoyltransferase (ArgF) and a gene encoding NCgl1221 which is a protein involved in glutamate export in the chromosome of the strain were deleted by homologous recombination so as to increase the intracellular content of glutamate which is a precursor of ornithine. Further, a gene encoding ornithine decarboxylase (ODC) derived from the wild-type E. coli W3110 which is involved in the synthesis of putrescine from ornithine was introduced into the chromosome of the strain. Furthermore, the own promoter of argCJBD gene cluster which codes for an enzyme involved in the synthesis of ornithine from glutamate was replaced by an improved promoter CJ7 promoter to prepare a Corynebacterium glutamicum strain having putrescine productivity. At this time, argCJBD encodes acetyl gamma glutamyl phosphate reductase (ArgC), acetylglutamate synthase or ornithine acetyltransferase (ArgJ), acetylglutamate kinase (ArgB), acetyl ornithine aminotransferase (ArgD) which are involved in the biosynthetic pathway of ornithine from glutamate. The Corynebacterium glutamicum strain having putrescine productivity thus prepared was deposited under the Budapest Treaty to the Korean Culture Center of Microorganisms (KCCM) on Nov. 24, 2010, with Accession No. KCCM11138P. A detailed description concerning the preparation of Corynebacterium sp. microorganism having putrescine productivity is given in Korean Patent Publication No. 10-2012-0064046, the disclosure of which is incorporated by reference is in its entirety.
A gene encoding acetyltransferase NCgl1469 in the Corynebacterium glutamicum KCCM11138P prepared in Reference Example 1 was weakened to produce no N-acetyl putrescine, thereby Corynebacterium glutamicum strain having improved putrescine productivity was prepared as another Corynebacterium sp. microorganism having putrescine productivity.
Specifically, based on the nucleotide sequence of the gene encoding NCgl1469 of Corynebacterium glutamicum ATCC13032, a pair of primers of SEQ ID NOs: 1 and 2 for obtaining a homologous recombination fragment of the N-terminal region of NCgl1469 and a pair of primers of SEQ ID NOs: 3 and 4 for obtaining a homologous recombination fragment of the C-terminal region of NCgl1469 were constructed as in the following Table 1.
PCR was performed using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and two pairs of primers so as to obtain PCR fragments of the N-terminal and C-terminal regions, respectively. These PCR fragments were electrophoresed to obtain the desired fragments. At this time, PCR reaction was carried out for 30 cycle of denaturation for 30 seconds at 95° C., annealing for 30 seconds at 55° C., and extension for 30 seconds at 72° C. The fragment of the N-terminal region thus obtained was treated with restriction enzymes, BamHI and SalI and the fragment of the C-terminal region thus obtained was treated with restriction enzymes, SalI and XbaI. The fragments thus treated were cloned into a pDZ vector treated with restriction enzymes, BamHI and XbaI, so as to construct a plasmid pDZ-NCgl1469(K/O).
The plasmid pDZ-NCgl1469(K/O) was transformed into Corynebacterium glutamicum KCCM11138P by electroporation to obtain a transformant. Then, the transformant was plated and cultured on BHIS plate (37 g/l of Braine heart infusion, 91 g/l of sorbitol, 2% agar) containing kanamycin (25 μg/ml) and X-gal (5-bromo-4-chloro-3-indolin-D-galactoside) for colony formation. From the colonies formed on the plate, blue-colored colonies were selected as the strain introduced with the plasmid pDZ-NCgl1469(K/O).
The selected strain was inoculated in CM medium (10 g/l of glucose, 10 g/l of polypeptone, 5 g/l of yeast extract, 5 g/l of beef extract, 2.5 g/l of NaCl, 2 g/l of urea, pH 6.8) and cultured with shaking at 30° C. for 8 hours. Subsequently, each cell culture was serially diluted from 10−4 to 10−10. Then diluted samples were plated and cultured on an X-gal-containing solid medium for colony formation.
From the colonies formed, the white colonies which appear at relatively low frequency were selected to prepare a Corynebacterium glutamicum strain having improved putrescine productivity by deletion of the gene encoding NCgl1469. The Corynebacterium glutamicum strain having improved putrescine productivity thus prepared was designated as KCCM11138P ΔNCgl1469 and deposited under the Budapest Treaty to the Korean Culture Center of Microorganisms (KCCM) on Dec. 26, 2011, with Accession No. KCCM11240P. A detailed description concerning the preparation of Corynebacterium sp. microorganism having putrescine productivity is given in Korean Patent Application No. 10-2012-0003634, the disclosure of which is incorporated by reference is in its entirety.
Corynebacterium glutamicum has no putrescine biosynthetic pathways. However, when Corynebacterium glutamicum is introduced with external ornithine decarboxylase to have an ability to produce putrescine, it produces and excretes putrescine extracellularly. It is indicated the presence of a transporter protein that functions as a passage of putrescine among numerous membrane proteins of Corynebacterium sp. microorganism.
In order to separate and isolate the putrescine exporter from the Corynebacterium sp. microorganism, a chromosome library of the wild-type Corynebacterium glutamicum ATCC13032 was prepared. Specifically, the chromosome of the Corynebacterium glutamicum ATCC13032 was treated with the restriction enzyme Sau3AI for incomplete cleavage. A gene fragment of 3˜5 kb was separated, and cloned into a pECCG122 vector treated with BamHI (shuttle vector of E. coli and Corynebacterium; Korean Patent Publication No. 10-1992-0000933).
The Corynebacterium chromosome library thus obtained was transformed into the putrescine-producing strain, Corynebacterium glutamicum KCCM11138P according to Reference Example 1, and then strains growing in 0.35 M putrescine-containing minimal medium (containing 10 g of glucose, 0.4 g of MgSO4.7H2O, 4 g of NH4Cl, 1 g of KH2PO4, 1 g of K2HPO4, 2 g of urea, 10 mg of FeSO4.7H2O, 1 mg of MnSO4.5H2O, 5 mg of nicotinamide, 5 mg of thiamine hydrochloride, 0.1 mg of biotin, 1 mM arginine, 25 mg of kanamycin, 0.35 M putrescine, based on 1 l of distilled water, pH 7.0) were selected. From about 5.5×105 transformants introduced with the Corynebacterium chromosome library, 413 colonies were selected, and then each library clone, of which putrescine resistance was also confirmed by secondary examination, was re-introduced into the putrescine-producing strain. Finally, one clone (B19), of which putrescine resistance was confirmed by tertiary examination, was selected. The clone was subjected to nucleotide sequence analysis. As a result, It was found to have NCgl2522 in B19 clone (
NCgl2522 which was isolated as the putrescine exporter from Corynebacterium glutamicum ATCC13032 has the amino acid sequence represented by SEQ ID NO: 21 which is encoded by a polynucleotide having the nucleotide sequence represented by SEQ ID NO: 20.
<2-1> Preparation of NCgl2522-Deleted Strain from ATCC13032-Based Putrescine-Producing Strain
In order to examine whether the Corynebacterium glutamicum ATCC13032-derived NCgl2522 is involved in putrescine export, a vector for deleting the gene encoding NCgl2522 was constructed.
Specifically, based on the nucleotide sequence of the gene encoding NCgl1469 which is represented by SEQ ID NO: 20, a pair of primers of SEQ ID NOs: 5 and 6 for obtaining a homologous recombination fragment of the N-terminal region of NCgl1469 and a pair of primers of SEQ ID NOs: 7 and 8 for obtaining a homologous recombination fragment of the C-terminal region of NCgl1469 were constructed as in the following Table 2.
PCR was performed using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and two pairs of primers so as to amplify PCR fragments of the N-terminal and C-terminal regions of NCgl2522 gene. These PCR fragments were electrophoresed to obtain the desired fragments. At this time, PCR reaction was carried out for 30 cycle of denaturation for 30 seconds at 95° C., annealing for 30 seconds at 55° C., and extension for 30 seconds at 72° C. The fragment of the N-terminal region thus obtained was treated with restriction enzymes, BamHI and SalI and the fragment of the C-terminal region thus obtained was treated with restriction enzymes, SalI and XbaI. The fragments thus treated were cloned into the pDZ vector treated with restriction enzymes, BamHI and XbaI, so as to construct a plasmid pDZ-1′NCgl2522(K/O).
The plasmid pDZ-1′NCgl2522(K/O) was transformed into Corynebacterium glutamicum KCCM11138P and KCCM11240P of Reference Examples 1 and 2 by electroporation, respectively so as to obtain transformants. Then, the transformants were plated and cultured on BHIS plate (37 g/l of Braine heart infusion, 91 g/l of sorbitol, 2% agar) containing kanamycin (25 μg/ml) and X-gal (5-bromo-4-chloro-3-indolin-D-galactoside) for colony formation. From the colonies formed on the plate, blue-colored colonies were selected as the strain introduced with the plasmid pDZ-1′NCgl2522(K/O).
The selected strains were cultured with shaking in CM medium (10 g/l of glucose, 10 g/l of polypeptone, 5 g/l of yeast extract, 5 g/l of beef extract, 2.5 g/l of NaCl, 2 g/l of urea, pH 6.8) at 30° C. for 8 hours. Subsequently, each cell culture was serially diluted from 10−4 to 10−10. Then, the diluted samples were plated and cultured on an X-gal-containing solid medium for colony formation. From the colonies formed, the white colonies which appear at relatively low frequency were selected to finally obtain strains in which the gene encoding NCgl2522 was deleted by secondary crossover. The strains finally selected were subjected to PCR using a pair of primers of SEQ ID NO: 5 and 8 to confirm deletion of the gene encoding NCgl2522. The Corynebacterium glutamicum mutant strains were designated as KCCM11138P ΔNCgl2522 and KCCM11240P ΔNCgl2522, respectively.
<2-2> Preparation of NCgl2522-Deleted Strain from ATCC13869-Based Putrescine-Producing Strain
NCgl2522—deleted strain was prepared from Corynebacterium glutamicum ATCC13869-based putrescine-producing strains, DAB12-a (argF deletion, NCgl1221 deletion, E. coli speC introduction, arg operon promoter substitution; see Reference Example 1) and DAB12-b (argF deletion, NCgl1221 deletion, E. coli speC introduction, arg operon promoter substitution, NCgl1469 deletion, see Reference Example 2) having the same genotype as KCCM11138P and KCCM11240P which are Corynebacterium glutamicum ATCC13032-based putrescine-producing strains.
Specifically, to examine the sequences of the gene encoding Corynebacterium glutamicum ATCC13869-derived NCgl2522 and the protein expressed therefrom, PCR was performed using the genomic DNA of Corynebacterium glutamicum ATCC13869 as a template and a pair of primers of SEQ ID NOs: 5 and 8. At this time, PCR reaction was carried out for 30 cycle of denaturation for 30 seconds at 95° C., annealing for 30 seconds at 55° C., and extension for 2 minutes at 72° C. The PCR product thus obtained was separated by electrophoresis, and subjected to sequencing. As a result, it was found that the nucleotide sequence of gene encoding Corynebacterium glutamicum ATCC13869-dervied NCgl2522 is represented by SEQ ID NO: 22, and the amino acid sequence of protein encoded thereby is represented by SEQ ID NO: 23. When the amino acid sequence of Corynebacterium glutamicum ATCC13032-derived NCgl2522 was compared to that of the Corynebacterium glutamicum ATCC13869-derived NCgl2522, they were found to have 98% sequence homology.
In order to delete the gene encoding Corynebacterium glutamicum ATCC13869-derived NCgl2522, in the same manner as in Example <2-1>, PCR was performed using the genomic DNA of Corynebacterium glutamicum ATCC13869 as a template and two pairs of primers of Table 2 so as to amplify PCR fragments of the N-terminal and C-terminal regions of NCgl2522 gene, respectively. These PCR fragments were electrophoresed to obtain the desired fragments. At this time, PCR reaction was carried out for 30 cycle of denaturation for 30 seconds at 95° C., annealing for 30 seconds at 55° C., and extension for 30 seconds at 72° C. The fragment of the N-terminal region thus obtained was treated with restriction enzymes, BamHI and SalI and the fragment of the C-terminal region thus obtained was treated with restriction enzymes, SalI and XbaI. The fragments thus treated were cloned into the pDZ vector treated with restriction enzymes, BamHI and XbaI, so as to construct a plasmid pDZ-2′NCgl2522(K/O).
In the same manner as in Example <2-1>, the plasmid pDZ-2′NCgl2522(K/O) was transformed into Corynebacterium glutamicum DAB12-a and DAB12-b, respectively. Strains, in which the gene encoding NCgl2522 was deleted, were selected. Corynebacterium glutamicum mutant strains thus selected were designated as DAB12-a ΔNCgl2522 and DAB12-b ΔNCgl2522, respectively.
<2-3> Evaluation of Putrescine Productivity of NCgl2522-Deleted Strain
In order to confirm the effect of NCgl2522 deletion on putrescine productivity in the putrescine-producing strain, putrescine productivities of the Corynebacterium glutamicum mutant strains prepared in Examples <2-1> and <2-2> were compared.
Specifically, 4 types of Corynebacterium glutamicum mutants (KCCM11138P ΔNCgl2522, KCCM11240P ΔNCgl2522, DAB12-a ΔNCgl2522, and DAB12-b ΔNCgl2522) and 4 types of parent strains (KCCM11138P, KCCM11240P, DAB12-a, and DAB12-b) were plated on CM plate media (1% glucose, 1% polypeptone, 0.5% yeast extract, 0.5% beef extract, 0.25% NaCl, 0.2% urea, 100 μl of 50% NaOH, 2% agar, pH 6.8, based on 1 L) containing 1 mM arginine, and cultured at 30° C. for 24 hours, respectively. 1 platinum loop of each strain thus cultured was inoculated in 25 ml of titer medium (8% Glucose, 0.25% soybean protein, 0.50% corn steep solids, 4% (NH4)2SO4, 0.1% KH2PO4, 0.05% MgSO4.7H2O, 0.15% urea, 100 g of biotin, 3 mg of thiamine hydrochloride, 3 mg of calcium-pantothenic acid, 3 mg of nicotinamide, 5% CaCO3, based on 1 L), and then cultured with shaking at 30° C. and 200 rpm for 98 hours. 1 mM arginine was added to the media for culturing all strains. The putrescine concentration in each culture was measured, and the results are shown in the following Table 3.
As shown in Table 3, a remarkable reduction in putrescine production was observed in 4 types of the NCgl2522—deleted Corynebacterium glutamicum mutant strains.
<3-1> Introduction of NCgl2522 into Transposon Gene in ATCC13032 Chromosome
In order to confirm high production of putrescine by additional chromosomal insertion of NCgl2522 gene (containing a self promoter region) in Corynebacterium sp. microorganism KCCM11138P having putrescine productivity, NCgl2522 was introduced into a transposon gene. A vector for transformation, pDZTn (Korean Patent Publication No. 10-2008-0033054) which allows introduction of the gene into a transposon gene on the chromosome of Corynebacterium sp. microorganism was used.
The NCgl2522 gene containing the self promoter was amplified using the chromosome of ATCC13032 strain as a template and a pair of primers of SEQ ID NO: 9 and 10 (see Table 4). At this time, PCR reaction was carried out for 30 cycle of denaturation for 30 seconds at 95° C., annealing for 30 seconds at 55° C., and extension for 30 seconds or 2 minutes at 72° C. Through PCR, a gene fragment having a size of 1.88 kb was obtained. This PCR product was electrophoresed in a 0.8% agarose gel to elute and purify a band of the desired size. pDZTn vector was treated with XhoI, and fusion cloning of the NCgl2522 PCR product of ATCC13032 strain was performed. In-FusionHD Cloning Kit (Clontech) was used in the fusion cloning. The resulting plasmid was designated as pDZTn-1′NCgl2522.
The plasmid pDZTn-1′NCgl2522 was transformed into Corynebacterium glutamicum KCCM11138P described in Reference Example 1 by electroporation to obtain transformants. From the transformants, a strain in which NCgl2522 was introduced into the transposon was selected in the same manner as in Example 2.
PCR was performed using genomic DNA of the selected strain and a pair of primers of SEQ ID NOs: 9 and 10 to confirm that NCgl2522 was introduced into the transposon by introduction of plasmid pDZTn-1′NCgl2522. At this time, PCR reaction was carried out for 30 cycle of denaturation for 30 seconds at 94° C., annealing for 30 seconds at 55° C., and extension for 2 minutes at 72° C.
A Corynebacterium glutamicum mutant strain thus selected was designated as KCCM11138P Tn: 1′NCgl2522.
<3-2> Preparation of NCgl2522 Promoter-Substituted Strain from ATCC13032-Based Putrescine-Producing Strain
In order to enhance NCgl2522 activity in the putrescine-producing strain, a CJ7 promoter (WO 2006/65095) was introduced in front of the NCgl2522 start codon on the chromosome.
First, a homologous recombination fragment containing a CJ7 promoter having the nucleotide sequence represented by SEQ ID NO: 24 and having the original NCgl2522 sequence at the both ends of the promoter was obtained. Specifically, PCR was performed using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and a pair of primers of SEQ ID NOs: 11 and 12 to obtain the 5′-terminal region of CJ7 promoter. At this time, PCR reaction was carried out for 30 cycle of denaturation for 30 seconds at 94° C., annealing for 30 seconds at 55° C., and extension for 30 seconds at 72° C. Further, PCR was performed using a pair of primers of SEQ ID NOs: 13 and 14 under the same conditions to obtain the CJ7 promoter region. Furthermore, PCR was performed using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and a pair of primers of SEQ ID NOs: 15 and 16 under the same conditions to obtain the 3′-terminal region of CJ7 promoter. The primers used in promoter substitution are the same as in the following Table 5.
Each PCR product thus obtained was fusion-cloned into pDZ vector treated with BamHI and XbaI. In-FusionHD Cloning Kit (Clontech) was used in the fusion cloning. The resulting plasmid was designated as pDZ-P(CJ7)-1′NCgl2522.
The plasmid pDZ-P(CJ7)-1′NCgl2522 thus prepared was transformed into Corynebacterium glutamicum KCCM11138P and KCCM11240P according to Reference Examples 1 and 2 by electroporation so as to prepare transformants. The transformants thus prepared were inoculated in CM media and cultured with shaking at 30° C. for 8 hours. Each cell culture obtained therefrom was diluted from 10′ to 10′, and plated and cultured on BHIS plate containing 25 μg/ml of kanamycin and X-gal for colony formation.
The white colonies appear at relatively low frequency, compared to majority of the colonies having blue color, and were selected to finally obtain a strain in which the NCgl2522 promoter was substituted with the CJ7 promoter by secondary crossover. PCR was performed using the genomic DNA of the selected strain as a template and a pair of primers of SEQ ID NOs: 13 and 16 to confirm that the CJ7 promoter was introduced in front of the NCgl2522 start codon on the chromosome by introduction of the plasmid pDZ1′CJ7(NCgl2522). At this time, PCR reaction was carried out for 30 cycle of denaturation for 30 seconds at 94° C., annealing for 30 seconds at 55° C., and extension for 1 minute at 72° C.
Corynebacterium glutamicum mutant strains thus selected were designated as KCCM11138P P(CJ7)-NCgl2522 and KCCM11240P P(CJ7)-NCgl2522, respectively.
<3-3> Introduction of NCgl2522 Gene into Transposon Gene on ATCC13869 Chromosome
In order to confirm high production of putrescine by additional chromosomal insertion of NCgl2522 gene in Corynebacterium glutamicum ATCC13869-derived putrescine strain, introduction of NCgl2522 (containing the promoter region) into a transposon gene was determined. NCgl2522 gene was amplified using the chromosome of ATCC13869 strain as a template and a pair of primers of SEQ ID NOs: 17 and 10 (see Table 6). At this time, PCR reaction was carried out for 30 cycle of denaturation for 30 seconds at 94° C., annealing for 30 seconds at 55° C., and extension for 30 seconds or 2 minutes at 72° C. Through PCR, a gene fragment having a size of 1.97 kb was obtained. The NCgl2522 PCR fragment thus prepared was fusion-cloned into pDZTn vector treated with XhoI. In-FusionHD Cloning Kit (Clontech) was used in the fusion cloning. The resulting plasmid was designated as pDZTn2′NCgl2522.
The plasmid pDZTn-2′NCgl2522 was transformed into Corynebacterium glutamicum DAB12-a in the same manner as in Example <3-1> to confirm introduction of NCgl2522 into the transposon.
A Corynebacterium glutamicum mutant strain thus selected was designated as DAB12-a Tn:2′NCgl2522.
<3-4> Preparation of NCgl2522 Promoter-Substituted Strain from ATCC13869-Based Putrescine-Producing Strain
In order to introduce the CJ7 promoter in front of the NCgl2522 start codon of Corynebacterium glutamicum ATCC13869, PCR was performed using the genomic DNA of Corynebacterium glutamicum ATCC13869 as a template and three pairs of primers given in the following Table 7 in the same manner as in Example <3-2>, respectively. Consequently, PCR fragments of the CJ7 promoter region, its N-terminal region and C-terminal region were amplified and then electrophoresed to obtain the desired fragments. At this time, PCR reaction was carried out for 30 cycle of denaturation for 30 seconds at 94° C., annealing for 30 seconds at 55° C., and extension for 30 seconds at 72° C. PCR fragments of the CJ7 promoter region, its N-terminal region and C-terminal region thus obtained were fusion-cloned into pDZ vector treated with BamHI and XbaI. In-FusionHD Cloning Kit (Clontech) was used in the fusion cloning. The resulting plasmid was designated as pDZ-P(CJ7)-2′NCgl2522.
The plasmid pDZ-′P(CJ7)-2′NCgl2522 was transformed into each of Corynebacterium glutamicum DAB12-a and DAB12-b in the same manner as in Example <3-2> to select strains, in which the CJ7 promoter was introduced in front of the NCgl2522 start codon. Corynebacterium glutamicum mutant strains thus selected were designated as DAB12-a P(CJ7)-NCgl2522 and DAB12-b P(CJ7)-NCgl2522.
<3-5> Evaluation of Putrescine Productivity of NCgl2522-Enhanced Strain
In order to confirm the effect of NCgl2522 activity enhancement by promoter substitution on putrescine productivity in the putrescine-producing strain, putrescine productivities of 6 types of Corynebacterium glutamicum mutant strains (KCCM11138P Tn:1′NCgl2522, KCCM11138P P(CJ7)-NCgl2522, KCCM11240P P(CJ7)-NCgl2522, DAB12-a Tn:2′NCgl2522, DAB12-a P(CJ7)-NCgl2522 and DAB12-b P(CJ7)-NCgl2522) prepared in Examples <3-1> to <3-4> and 4 types of parent strains (KCCM11138P, KCCM11240P, DAB12-a and DAB12-b) were compared. Each strain was cultured in the same manner as in Example 2-3, and the putrescine concentration in each culture was measured, and the results are shown in the following Table 8.
As shown in Table 8, an increase in putrescine production was observed in all 6 types of Corynebacterium glutamicum mutant strains in which NCgl2522 activity was enhanced by additional introduction of NCgl2522 into the transposon or by promoter substitution.
In order to confirm that intracellular putrescine concentration is reduced by an enhancing ability to export putrescine in the Corynebacterium glutamicum mutant strain having enhanced NCgl2522 activity, intracellular putrescine concentrations in Corynebacterium glutamicum mutant strain KCCM11138P Tn:1′NCgl2522 and in parent strain KCCM11138P were measured by extraction using an organic solvent. Intracellular metabolite analysis was carried out in accordance with a method described in the literature (Nakamura J et al., Appl. Environ. Microbiol. 73(14): 4491-4498, 2007).
First, Corynebacterium glutamicum mutant strain KCCM11138P Tn:1′NCgl2522 and parent strain KCCM11138P were inoculated in 25 ml of CM liquid media (1% glucose, 1% polypeptone, 0.5% yeast extract, 0.5% beef extract, 0.25% NaCl, 0.2% urea, 100 l of 50% NaOH, pH 6.8, based on 1 L) containing 1 mM arginine, and cultured with shaking at 30° C. and 200 rpm. When cell growth reached exponential phase during cultivation, cells were isolated from the culture media by rapid vacuum filtration (Durapore HV, 0.45 m; Millipore, Billerica, Mass.). The cell-adsorbed filter was washed with 10 ml of cooled water twice, and then dipped in methanol containing 5 M morpholine ethanesulfonic acid and 5 M methionine sulfone for 10 minutes.
The extraction liquid obtained therefrom was mixed well with an equal volume of chloroform and 0.4-fold volume of water, and the aqueous phase was only applied to a spin column to remove protein contaminants. The filtered extraction liquid was analyzed by capillary electrophoresis mass spectrometry, and the results are shown in the following Table 9.
As shown in Table 9, a reduction in the intracellular putrescine concentration was observed in Corynebacterium glutamicum mutant strain KCCM11138P Tn:1′NCgl2522 having enhanced NCgl2522 activity, compared to parent strain KCCM11138P, It suggests that an imporved ability to export putrescine by enhancement of NCgl2522 activity in Corynebacterium glutamicum mutant strain KCCM11138P Tn:1′NCgl2522, leads to effective extracellular export of intracellular putrescine.
In order to examine the effect of NCgl2522 on putrescine resistance, putrescine resistance of KCCM11240P, KCCM11240P ΔNCgl2522, and KCCM11240P P(CJ7)NCgl2522 strains was evaluated.
Each strain was inoculated in 2 ml of CM liquid containing 1 mM arginine medium and cultured at 30° C. for about 10 hours, followed by dilution in this order of 105, 104, 103, 102 and 101. Each dilution thus prepared was spotted on 0 M or 0.8 M putrescine-containing CMA plate (1% glucose, 1% polypeptone, 0.5% yeast extract, 0.5% beef extract, 0.25% NaCl, 0.2% urea, 1.8% agar, 1 mM arginine, pH 6.8, based on 1 L) and then cultured at 30° C. for 48 hours to compare growth differences between strains.
As a result, the strains showed two different growth patterns. As shown in
In order to confirm whether putrescine production is increased when NCgl2522 of Corynebacterium glutamicum ATCC13032 is expressed in the wild-type E. coli strain W3110 having a putrescine biosynthetic pathway, a vector expressing speC which is a putrescine synthetic enzyme or a vector expressing NCgl2522 were introduced into W3110.
In order to prepare the speC-expressing vector, W3110 chromosome as a template and a pair of primers of SEQ ID NOs: 34 and 35 were used to amplify a speC gene fragment of about 2.1 kb (see Table 10). This PCR product was electrophoresed in a 0.8% agarose gel, and then a band of the desired size was eluted and purified. pSE280 vector (Invitrogen) containing Trc promoter was treated with NcoI and EcoRI, and then the speC PCR product was fusion-cloned into this vector. In-Fusion® HD Cloning Kit (Clontech) was used in the fusion cloning. The resulting plasmid was designated as pSE280-speC.
In order to prepare the NCgl2522-expressing vector, pSE280 as a template and a pair of primers of SEQ ID NOs: 36 and 37 were used to obtain a Trc promoter fragment, and Corynebacterium glutamicum ATCC13032 chromosome as a template and a pair of primers of SEQ ID NOs: 38 and 39 were used to obtain an NCgl2522 fragment. These PCR products were electrophoresed in a 0.8% agarose gel, and then bands of the desired size were eluted and purified. The trc promoter fragment and the NCgl2522 fragment were fusion-cloned into pcc1BAC treated with HindIII. The resulting plasmid was designated as pcc1BAC-P(trc)NCgl2522.
The plasmids, pSE280-speC or pcc1BAC-P(trc)-NCgl2522, were transformed into W3110. Transformation into E. coli was carried out using 2×TSS solution (Epicentre). pSE280-speC-introduced E. coli was plated and cultured on an ampicillin (100 μg/ml)containing LB plate (10 g of Tryptone, 5 g of yeast extract, 10 g of Nacl, 2% agar, based on 1 l) for colony formation. pcc1BAC-P(trc)-NCgl2522-introduced E. coli was plated and cultured on a chloramphenicol (35 μg/ml)-containing LB plate for colony formation. Putrescine productivities of the strains thus obtained were examined.
Specifically, W3110, W3110 pSE280-speC, and W3110 pcc1BAC-P(trc)-NCgl2522 were inoculated on LB, LA and LC plates, respectively and cultured at 37° C. for 24 hours, and then inoculated in 25 ml of titer medium (2 g of (NH4)2PO4, 6.75 g of KH2PO4, 0.85 g of citric acid, 0.7 g of MgSO4.7H2O, 0.5% (v/v) trace element, 10 g of glucose, 3 g of AMS, 30 g of CaCO3, based on 1 L) and cultured at 37° C. for 24 hours. A trace metal solution contains 5 M HCl, 10 g of FeSO4.7H2O, 2.25 g of ZnSO4.7H2O, 1 g of CuSO4.5H2O, 0.5 g of MnSO4.5H2O, 0.23 g of Na2B4O7.10H2O, 2 g of CaCl2.2H2O, and 0.1 g of (NH4)6Mo7O2.4H2O in 1 L.
The putrescine concentration in each culture was measured, and the results are shown in the following Table 11.
As shown in Table 11, high putrescine production was observed in the W3110 pcc1 BAC-P(trc)-NCgl2522 strain introduced with NCgl2522, compared to W3110pcc1BACpSE280-speC strain introduced with the putrescine biosynthetic enzyme, speC.
This result demonstrates that NCgl2522 protein also has the ability to export putrescine in E. coli.
The present inventors found that additional introduction of NCgl2522 into the transposon of Corynebacterium sp. microorganism KCCM11138P having putrescine productivity was performed to enhance NCgl2522 activity of Corynebacterium glutamicum strain, and thus putrescine could be produced in a high yield owing to the increased ability to export putrescine, and they designated the strain as Corynebacterium glutamicum CC01-0510, and deposited under the Budapest Treaty to the Korean Culture Center of Microorganisms (KCCM) on Mar. 8, 2013, with Accession No. KCCM11401P.
Based on the above description, it should be understood by those skilled in the art that other specific embodiments may be employed in practicing the invention without departing from the technical idea or essential features of the invention. In this regard, the above-described examples are for illustrative purposes only, and the invention is not intended to be limited by these examples. The scope of the present invention should be understood to include all of the modifications or modified form derived from the meaning and scope of the following claims or its equivalent concepts, rather than the above detailed description.
A Corynebacterium sp. microorganism having improved putrescine productivity of the present invention is modified to have enhanced NCgl2522 activity of exporting intracellular putrescine, compared to its endogenous activity, resulting in increased extracellular export of putrescine and increased putrescine resistance.
Further, when NCgl2522 was expressed in E. coli containing a putrescine synthetic pathway of the present invention, the amount of extracellular putrescine was found to increase. Accordingly, Corynebacterium glutamicum-derived NCgl2522 can be applied to a microorganism having putrescine productivity, which can be widely used in the effective production of putrescine.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2013-0030020 | Mar 2013 | KR | national |
| 10-2014-0017243 | Feb 2014 | KR | national |
This application is a continuation of U.S. application Ser. No. 14/778,383, filed Sep. 18, 2015 (now issued U.S. Pat. No. 10,221,433), which is the National Stage of International Application No. PCT/KR2014/001509, filed Feb. 25, 2014, which claims priority to Korean Patent Application No. 10-2014-0017243, filed Feb. 14, 2014 and Korean Patent Application No. 10-2013-0030020, filed Mar. 20, 2013. These applications are incorporated herein by reference in their entireties.
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| Number | Date | Country | |
|---|---|---|---|
| 20190136274 A1 | May 2019 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14778383 | US | |
| Child | 16246082 | US |
