WO2010101359A2 - L-아미노산 생산 미생물 및 이를 이용한 l-아미노산 생산 방법 - Google Patents
L-아미노산 생산 미생물 및 이를 이용한 l-아미노산 생산 방법 Download PDFInfo
- Publication number
- WO2010101359A2 WO2010101359A2 PCT/KR2010/000932 KR2010000932W WO2010101359A2 WO 2010101359 A2 WO2010101359 A2 WO 2010101359A2 KR 2010000932 W KR2010000932 W KR 2010000932W WO 2010101359 A2 WO2010101359 A2 WO 2010101359A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- sucrose
- microorganism
- amino acid
- seq
- escherichia
- Prior art date
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K14/26—Klebsiella (G)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
Definitions
- the present invention encodes a sucrose metabolizing enzyme derived from sucrose magnetizing microorganisms in a sucrose non-assimilative Escherichia sp.
- Microorganism having L-amino acid producing ability and sucrose phosphoenolypyruvate dependent sucrose phosphotransferase system (PTS) activity.
- the present invention relates to an Escherichia genus microorganism having sucrose magnetization and L-amino acid producing ability obtained by introducing a gene, and a method for producing L-amino acid using the same.
- Starch sugar which is mainly used in the fermentation industry, is rapidly increasing in demand due to the recent accelerated development of biofuels. Therefore, when molasses containing a large amount of sucrose or sucrose, which is cheaper than starch sugar, is used as a carbon source of the fermentation industry, higher cost competitiveness can be secured.
- sucrose magnetization In order to give sucrose magnetization to industrial E. coli, a method of introducing a sucrose soluble gene or a gene group derived from a microorganism having sucrose magnetization is mainly used.
- enterobacteriacea Enterobacteriaceae
- Salmonella in the family Salmonella
- Bell J. Bacteriol. (1982) 151: 68-76, Mol. Microbiol. (1988) 2: 1-8, J. Bacteriol. (1991) 173: 7464-7470, US Patent No. 7179623
- Klebsiella pneumoniae Klebsiella pneumoniae
- the present inventors have found that a microorganism of the L-amino acid producing Escherichia genus, in which a sucrose utilizing gene group derived from Klebsiella pneumoniae having sucrose magnetization, is introduced, produces L-amino acid in high yield from sucrose.
- the present invention has been completed.
- An object of the present invention is to provide sucrose magnetization to the sucrose non-magnetizing Escherichia spp. Microorganisms having L-amino acid production capacity, to provide an Escherichia microorganism having sucrose magnetization and L-amino acid production capacity.
- the present invention is sucrose to the sucrose non-assimilative Escherichia genus microorganism having L-amino acid production capacity and sucrose phosphoenolypyruvate dependent sucrose phosphotransferase system (PTS) activity
- a microorganism of the genus Escherichia obtained by introducing a gene encoding a sucrose metabolizing enzyme derived from a chemical conversion microorganism having sucrose magnetization and L-amino acid production ability.
- sucrose non-assimilative microorganism means a microorganism that does not use sucrose as a carbon source
- microorganism having sucrose magnetization and L-amino acid production ability refers to a L- It means a microorganism capable of producing amino acids.
- sucrose metabolizing enzyme in the present invention means an enzyme required for using sucrose as a carbon source, fructokinase (fructokinase), sucrosre porin, sucrosre PTS permease, sucrose hydrola Sucrose hydrolase, invertase, and the like, but are not limited thereto.
- sucrose metabolizing enzyme is also referred to as "Scr-PTS enzyme”.
- the metabolic system which can use sucrose as a carbon source, is largely a phosphoenolpyruvate dependent sucrose phosphotransferase (PTS) -based sucrose metabolism system (Scr-PTS), depending on a source of phosphate for phosphorylation of sucrose introduced into a cell. System) and a non-PTS based sucrose metabolic system (Scr-non PTS system). Most microorganisms that can utilize sucrose have an Scr-PTS system.
- PTS sucrose phosphotransferase
- Scr-PTS sucrose metabolism system
- Most microorganisms that can utilize sucrose have an Scr-PTS system.
- a representative example of a PTS-based Scr-PTS system using phosphoenolpyruvate (PEP) as a source of phosphate for the phosphorylation of sucrose is the conjugation plasmid pUR400 of Salmonella typhmurium , a Gram-negative microorganism.
- keulrep City include Ella pneumoniae scr Les gyulron present on the chromosome of (Klebsiella pneumoniae).
- scr regulators include scrK ( fructokinase ), scrY (sucrose porin), scrA (sucrose-specific EIIBC component), scrB (sucrose-6-phosphate hydrolase) and scrR (LacI-related sucrose-specific refresher ) And two operons, scrK and scrYAB, are negatively regulated by the ScrR repressor ( Mol. Microbiol. (1993) 9: 195-209).
- ScrR repressor Mol. Microbiol. (1993) 9: 195-209.
- external sucrose enters the periplasmic space through ScrY, an outer membrane protein (OMP), and the imported sucrose is a sucrose PTS circuit containing ScrA.
- OMP outer membrane protein
- Sucrose-6-phosphate is hydrolyzed to glucose-6-phosphate and fructose by ScrB, and fructose is converted to fructose-6-phosphate by ATP dependent ScrK to be combined with glucose-6-phosphate Metabolized throughout the process ( J. Biotechnol. (2001) 92: 133-158).
- the sucrose PTS circuit which acts to transport sucrose into cells in the form of sucrose-6-phosphate, includes enzyme I (EI), histidine protein (HPr), and glucose-specific enzyme IIA (EIIA crr Glc , enzyme IIA) and sucrose-specific enzyme IIBC EIIBC scr , enzyme IIBC) ( J. Biotechnol. (2001) 92: 133-158 / J. Biotechnol. (2004) 110: 181-199).
- the Scr-PTS system of Gram-positive bacteria is the scr regulator of Streptococcus mutans and consists of the genes scrK , scrA , scrB , and scrR ( J. Bacteriol . (2003) 185: 5791-5799).
- the csc regulator is well known as the Scr-non PTS system, a sucrose metabolism system not based on PTS for the influx of sucrose into cells.
- csc regulators are mainly derived from Escherichia coli with sucrose magnetization, for example, csc regulators of EC3132 ( Mol. Gen. Genet. (1992) 235: 22-32, US Patent No. 6960455) of wild type E. coli . , Escherichia coli KO11 csc regulator ( Biotechnol. Lett. (2004) 26: 689-693), csc regulator of pathogenic E. coli O157: H7 (J. Bacteriol.
- csc regulators consist of cscB (proton symport-type sucrose permease ), cscK ( fructokinase ), cscA (sucrose hydrolase) and cscR (LacI-related sucrose-specific refresher)
- cscB proton symport-type sucrose permease
- cscK fructokinase
- cscA fructokinase
- cscA sucrose hydrolase
- cscR LacI-related sucrose-specific refresher
- Scr-non PTS systems have the disadvantage that they cannot efficiently utilize low concentrations of sucrose. Escherichia coli introduced with the csc regulator has been reported to have a doubling time of 20 hours in a medium containing less than 0.2% sucrose ( J. Bacteriol. (2002) 184: 5307-5316). However, unlike the Scr-non PTS system, the Scr-PTS system can efficiently enter a low concentration of sucrose into the cell. The reason is that when ScrA, which acts as sucrose PTS permease in the Scr-PTS system, enters into the cell extracellular sucrose, phosphoenolpyruvate (PEP) is converted into pyruvate, resulting in free phosphate.
- PEP phosphoenolpyruvate
- sucrose PTS circuit composed of enzyme I, histidine protein, and glucose specific enzyme IIA, and serves to convert to sucrose-6-phosphate.
- sucrose is introduced into cells using PEP used as an energy source, thus efficiently introducing low sucrose into cells. can do.
- the CscB of Scr-non PTS which introduces external sucrose into cells by the actual concentration gradient of hydrogen, has a Km value of 1.0 mM ( Biochem. Biophys Res. Commun. (1995) 208: 1116-1123), while ScrA The Km value of 10 ⁇ m ( J. Bacteriol.
- the Scr-PTS system contains ScrY, a protein that allows efficient use of low concentrations of sucrose.
- ScrY acts as a sucrose porin to induce extracellular sucrose into the extracellular matrix and is known to significantly reduce sucrose transport when sucrose porin is not normally expressed ( J. Bacteriol. (1991) 173: 449-456. ). That is, in the Scr-PTS system, ScrY can introduce external sucrose into the extracellular matrix, and the imported sucrose can be introduced into the cell at a high speed through the PTS system, so that it can efficiently utilize low sucrose.
- the gene encoding the sucrose metabolic enzyme derived from sucrose magnetizable microorganism is a gene derived from a microorganism having sucrose magnetization, preferably from a microorganism having a Scr-PTS system based on PTS It is a gene.
- sucrose chair gene encoding a sucrose-metabolizing enzymes of the converted microbial refers to a gene derived from sucrose chair Mars keulrep when Ella (Klebsiella) in micro-organisms or air Winiah (Erwinia) in the microorganism, and more preferably Preferably it may be a gene derived from Klebsiella pneumoniae ATCC700721 or Erwinia carotovora ATCCBAA-672.
- the gene encoding the sucrose metabolizing enzyme derived from sucrose magnetizing microorganisms is fructokinase, sucrose porin, sucrose PTS permease, sucrose hydrolase from Klebsiella pneumoniae May be a combination consisting of genes encoding an aze and sucrose transcriptional regulators.
- the genes encoding the fructokinase , sucrose porin, sucrose PTS permease , sucrose hydrolase and sucrose transcriptional regulators from sucrose magnetizable microorganisms are scrK of SEQ ID NO: 6, SEQ ID NO: 7, respectively. in may be scrY, SEQ ID NO: 8 of scrA, SEQ ID NO: 9 of scrB, and scrR of SEQ ID NO: 10.
- the sucrose non-magnetic E. coli microorganisms include enzyme I (EI, Enzyme I), histidine protein (HPr) and glucose-specific enzyme IIA (EIIA crr Glc , enzyme) excluding ScrA.
- enzyme I EI, Enzyme I
- HPr histidine protein
- EIIA crr Glc glucose-specific enzyme IIA
- the gene encoding enzyme I ptsI , SEQ ID NO: 19
- the gene encoding histidine protein ptsH , SEQ ID NO: 20
- the gene encoding glucose-specific enzyme IIA in a sucrose Escherichia spp . crr , SEQ ID NO: 21 should be normally expressed.
- sucrose porin sucrose PTS permease derived from sucrose magnetizing microorganisms, in the sucrose non-magnetizing Escherichia spp.
- Microorganism for the production of sucrose magnetizing and L-amino acid producing ability according to the present invention Introduction of genes encoding sucrose hydrolase, fructokinase, and sucrose transcriptional regulators can be performed by a variety of methods well known in the art.
- a recombinant vector is constructed by introducing a nucleotide sequence encoding sucrose porin, sucrose PTS permease, sucrose hydrolase, fructokinase, and sucrose transcriptional regulator into a vector to construct a recombinant vector, and into the constructed recombinant vector.
- the vector usable for obtaining the Escherichia genus microorganism of the present invention is not particularly limited and known expression vectors can be used.
- pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, or pMW118 vector may be used.
- transformation means introducing a gene into a host cell so that it can be expressed in the host cell. If the transformed gene can be expressed in the host cell, it may be inserted into the chromosome of the host cell in the cell, or may exist independently of the chromosome.
- transgenes are defined as polynucleotides capable of encoding a polypeptide and include DNA and RNA. The transgene may be in a suitable form that can be introduced into and expressed in a host cell. For example, the transgene may be introduced into a host cell in the form of an expression cassette, which is a polynucleotide construct containing all the elements necessary for its expression.
- Expression cassettes typically include a promoter, transcription termination signal, ribosomal binding site and translation termination signal operably linked to a transgene.
- the expression cassette may be in the form of an expression vector capable of self replication.
- the transgene may be introduced into the host cell in its own form or in the form of a polynucleotide structure, and may be operably linked to a sequence required for expression in the host cell.
- the sucrose non-magnetic non-Escaperia microorganism having the ability to produce L-amino acid comprises a gene encoding the Scr-PTS enzyme from Klebsiella pneumoniae to obtain sucrose magnetization Can be transformed by recombinant vectors.
- the sucrose non-magnetic non-Escaperia microorganism having L-amino acid producing ability can be transformed by a recombinant plasmid comprising the sequence of SEQ ID NO: 17 to obtain sucrose magnetization.
- SEQ ID NO: The recombinant plasmid of 17 keulrep when Ella pneumoniae derived scrKYABR from (ATCC700721), i.e., fructo of SEQ ID NO: 6 encoding the kinase scrK, of SEQ ID NO: 7 encoding a sucrose Foreign scrY, sucrose ScrA of SEQ ID NO: 8 encoding a PTS permease , scrB of SEQ ID NO: 9 encoding a sucrose hydrolase , and scrR of SEQ ID NO: 10 encoding a sucrose transcriptional regulator.
- Escherichia microorganisms having sucrose magnetization and L-amino acid producing ability according to the present invention can produce L-amino acid, while at the same time sucrose porin, sucrose PTS permease, sucrose hydrolase, fructokinase and sucrose transcription It is a microorganism belonging to the genus Escherichia that retains the activity of the regulator and maintains the activity of the sucrose PTS cycle, and may preferably be Escherichia coli .
- the L-amino acid is L-threonine, O-succinyl-homoserine, O-acetyl-homoserine, L-methionine, L-lysine, L-homoserine, L-isoleucine , L-valine, or L-tryptophan.
- the L-amino acid may be L-threonine.
- Escherichia spp. Microorganisms having sucrose magnetization and L-amino acid production capacity have E. coli ABA5G having L-threonine production capacity having the nucleotide sequence of SEQ ID NO: 17 including the scrKYABR gene group.
- E. coli CA03-0207 (KCCM 10993) obtained by transformation with a vector.
- the present invention comprises the steps of culturing an Escherichia microorganism having sucrose magnetization and L-amino acid production capacity in a medium containing sucrose as a carbon source, and recovering L-amino acid from the culture medium, L Provides a method for producing amino acids.
- the L-amino acid is L-threonine, O-succinyl-homoserine, O-acetyl-homoserine, L-methionine, L-lysine, L-homoserine, L-isoleucine , L-valine, or L-tryptophan.
- the L-amino acid may be L-threonine.
- the method for producing L-amino acid according to the present invention includes culturing an Escherichia genus microorganism having sucrose magnetization and L-amino acid producing ability.
- the step of culturing the microorganism of the genus Escherichia may be carried out in a medium and culture conditions suitable for the microorganism.
- a medium and culture conditions suitable for the microorganism Those skilled in the art to which the present invention pertains can easily select and adjust the medium and culture conditions appropriate for a given strain.
- Culture methods include, but are not limited to, batch, continuous and fed-batch cultures.
- the step of culturing the E. coli microorganism is carried out by controlling the temperature, pH and the like under aerobic conditions in a conventional medium containing a suitable carbon source, nitrogen source, amino acids, vitamins, etc., including the sucrose Incubate.
- the medium used in the present invention includes a sucrose or sucrose as a primary sugar source containing a large amount of sucrose, and may include various carbon sources in addition to the main carbon source in an appropriate amount.
- Nitrogen sources that may be included in the medium include organic nitrogen sources and urea such as peptone, yeast extract, gravy, malt extract, corn steep liquor, and soybean wheat, ammonium sulfate, ammonium phosphate, ammonium phosphate, and anmonium carbonate that can be used alone or in combination
- inorganic nitrogen sources such as ammonium nitrate.
- Sources of phosphorus in the medium may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate or sodium-containing salts thereof.
- amino acids, vitamins and appropriate precursors may be included.
- the main components of the medium may be added batchwise or continuously to the culture.
- Incubation temperature may be usually 27 °C to 37 °C, preferably 30 °C to 35 °C. The culturing can be continued as long as the amount of production of the desired substance L-amino acid is increased under given conditions, for example, it can be 10 to 100 hours.
- the method for producing L-amino acid according to the present invention includes recovering L-amino acid from a culture of a microorganism. Recovery of L-amino acids from the culture can be carried out using any suitable method known in the art, depending on the culture method, for example, batch, continuous or fed-batch culture method.
- L-amino acid producing microorganisms having sucrose magnetization according to the present invention, it is possible to economically produce L-amino acids using cheap sucrose as a carbon source.
- FIG. 1 is a schematic of recombinant plasmid pAscrKP comprising scrKYABR from Klebsiella pneumoniae (ATCC700721) according to one embodiment of the invention.
- sucrose soluble gene group scr regulatoryon was obtained from the sucrose magnetizing microorganism.
- scr regulators include scrK ( fructokinase ), scrY (sucrose porin), scrA (sucrose-specific EIIBC component), scrB (sucrose-6-phosphate hydrolase) and scrR (LacI-related sucrose-specific refresher ), Two operons, scrK and scrYAB, are negatively regulated by the ScrR repressor ( Mol. Microbiol. (1993) 9: 195-209).
- ScrKYABR a scr regulator of Klebsiella pneumoniae, uses primer pairs of SEQ ID NO: 1 and SEQ ID NO: 2, and scrKYABR of Erwinia Carobora , through PCR using primer pairs of SEQ ID NO: 3 and SEQ ID NO: 4 All five genes, each present continuously on the genome, were obtained by amplification with a single polynucleotide.
- the primers of SEQ ID NOs: 1 and 3 have restriction enzyme ApaLI sites, and the primers of SEQ ID NOs: 2 and 4 have StuI sites.
- the primers used were information on the scrKYABR and surrounding sequences of Klebsiella pneumoniae (KEGG organism, kpn) and Erwinia carotobora (KEGG) registered in the Kyoto Encyclopedia of Genes and Genomes (KEGG). Based on the production.
- PCR was repeated at 25 ° C. after 3 minutes of denaturation at 94 ° C., followed by 25 cycles of 30 second denaturation at 94 ° C., 30 second annealing at 56 ° C., and 5 minute polymerization at 72 ° C., followed by 72 ° C.
- the polymerization was carried out for 7 minutes at.
- 7046 bp of polynucleotide was obtained from Klebsiella pneumoniae and 7223 bp of polynucleotide was obtained from Erwinia Carotobora.
- Each polynucleotide obtained by PCR was treated with ApaLI and FspI , and then cloned into the ApaLI and FspI positions of the pACYC177 vector, transformed into Escherichia coli DH5 ⁇ , and containing 1% sucrose (MacConkey agar plate). ). The dark purple colonies were selected from the colonies formed, and plasmids derived from Klebsiella pneumoniae and Erwinia carotobora were obtained by using a commonly known plasmid miniprep method. .
- Klebsiella pneumoniae obtained in the above (1) scr With regularon Named the plasmid pAscrKP, using the sequencing method commonly used in the technical field to which the present invention belongs Apali , FspI Cloned into position scrKYABR The base sequence of was determined (SEQ ID NO: 5). 1 is derived from Klebsiella pneumoniae (ATCC700721) scrKYABR The construction of the recombinant plasmid pAscrKP comprising a.
- SEQ ID NO: 5 scrKYABR
- the gene SEQ ID NO: 6
- the 1395th to the 2912th nucleotide sequences scrY The gene (SEQ ID NO: 7), the nucleotide sequence of 3017 to 4387 scrA
- the gene SEQ ID NO: 8
- the nucleotides 4387 to 5787 scrB Gene SEQ ID NO: 9
- the 5817-6821 nucleotide sequences scrR The gene (SEQ ID NO: 10) was identified.
- the plasmid is named pAscrEC, using a sequencing method commonly used in the technical field to which the present invention belongs.
- Apali FspI Cloned into position scrKYABR DNA sequencing was determined (SEQ ID NO: 11).
- SEQ ID NO: 11 scrKYABR
- SEQ ID NO: 12 The 412th to 1347th nucleotide sequence in sequence scrK
- the gene SEQ ID NO: 12
- the 15th through 3073 nucleotide sequences scrY Gene SEQ ID NO: 13
- the 3153rd to 4523rd nucleotide sequences scrA The gene (SEQ ID NO: 14), the 4523th to 5932th nucleotide sequences scrB Gene (SEQ ID NO: 15), and the 5963-6982 nucleotide sequences scrR It was confirmed by the gene (SEQ ID NO: 16).
- E. coli ABA5G transformed with pAscrKP or pAscrEC was plated on a MacConkey agar plate containing 1% sucrose, and dark purple colonies were selected from the colonies formed. PCR was confirmed for the selected colonies to have a plasmid containing the sucrose availability gene.
- the colonies obtained in (1) were incubated overnight in an LB solid medium (tryptone 1 g, NaCl 1 g, yeast extract 0.5 g / 100 ml, 1.5% agar) in a 33 ° C. incubator, and are shown in Table 1 below.
- One platinum was inoculated into 25 mL titer medium containing sucrose having a composition as a main carbon source, followed by incubation for 70 hours in an incubator at 33 ° C. and 200 rpm.
- the control group was E. coli ABA5G, a parent strain that was not transformed with plasmids, and culture was also performed in a medium of the composition of Table 1 in which sucrose was substituted with glucose, in order to compare the utilization rate and threonine productivity of sucrose relative to glucose.
- the culture results are summarized in Table 2 below.
- E. coli ABA5G / pAscrEC containing pAscrEC produced 44.3 g / L (without data) sucrose and produced 12.2 g / L of L-threonine and E. coli ABA5G / pAscrKP containing pAscrKP. 70% of sucrose was used in the culture medium for 70 hours, and 26.5 g / L of L-threonine was produced. It was confirmed that E. coli ABA5G containing pAscrEC or pAscrKP showed sucrose availability, while E. coli ABA5G, a parent strain containing no plasmid, was not used at all.
- ABA5G with pAscrKP was found to have better sucrose availability and threonine productivity than ABA5G with pAscrEC.
- ABA5G containing pAscrKP had an L-threonine concentration of 21.2 g / L in glucose-containing titer medium, whereas L-threonine concentration of 26.5 g / L was determined in a titer medium containing sucrose.
- Threonine productivity was found to increase about 1.3 times.
Abstract
Description
조성물 | 농도 (리터당) |
수크로오스 | 70 g |
KH2PO4 | 2 g |
(NH4)2SO4 | 25 g |
MgSO4·7H2O | 1 g |
FeSO4·7H2O | 5 mg |
MnSO4·4H2O | 5 mg |
효모 추출물 | 2 g |
탄산칼슘 | 30 g |
pH | 6.8 |
글루코오스 (70 g/L) | 수크로오스 (70 g/L) | |||
OD | L-쓰레오닌(g/L) | OD | L-쓰레오닌(g/L) | |
ABA5G | 14.7 | 21.5 | - | - |
ABA5G/pAscrEC | 14.4 | 21.6 | 8.4 | 12.2 |
ABA5G/pAscrKP | 14.6 | 21.2 | 15.0 | 26.5 |
Claims (8)
- L-아미노산 생산능 및 수크로오스 PTS(phosphoenolypyruvate dependent sucrose phosphotransferase system) 활성을 갖는, 수크로오스 비자화성(non-assimilative) 에스케리시아(Escherichia) 속 미생물에 클렙시엘라 뉴모니애(Klebsiella pneumoniae)에서 유래된 프럭토키나아제, 수크로오스 포린, 수크로오스 PTS 퍼미아제, 수크로오스 히드롤라아제, 및 수크로오스 전사 조절자를 코딩하는 유전자를 도입하여 수득되고, 상기 수크로오스 PTS는 효소 I(EI), 히스티딘 단백질(Histidine protein), 및 글루코오스 특이적 효소 IIA(EIIAcrr Glc , EII)로 구성된 것인, 수크로오스 자화성 및 L-아미노산 생산능을 갖는 에스케리시아 속 미생물.
- 제1항에 있어서, 상기 프럭토키나아제, 수크로오스 포린, 수크로오스 PTS 퍼미아제, 수크로오스 히드롤라아제 및 수크로오스 전사 조절자를 코딩하는 유전자는 각각 서열번호 6의 scrK, 서열번호 7의 scrY, 서열번호 8의 scrA, 서열번호 9의 scrB, 및 서열번호 10의 scrR인 것인 에스케리시아 속 미생물.
- 제1항에 있어서, 상기 에스케리시아 속 미생물은 상기 수크로오스 비자화성 에스케리시아 속 미생물을 서열번호 17의 재조합 벡터로 형질전환시켜 수득된 것인 에스케리시아 속 미생물.
- 제1항에 있어서, 상기 에스케리시아 속 미생물은 대장균(E. coli)인 것인 에스케리시아 속 미생물.
- 제4항에 있어서, 상기 대장균은 대장균 CA03-0207(KCCM 10993)인 것인 에스케리시아 속 미생물.
- 제1항에 있어서, 상기 L-아미노산은 L-쓰레오닌인 것인 에스케리시아 속 미생물.
- 제1항 내지 제6항 중 어느 한 항에 따른 에스케리시아 속 미생물을 수크로오스를 탄소원으로 포함하는 배지에서 배양하는 단계, 및상기 배양액으로부터 L-아미노산을 회수하는 단계를 포함하는, L-아미노산을 생산하는 방법.
- 제7항에 있어서, 상기 L-아미노산은 L-쓰레오닌인 것인 방법.
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP10748903.1A EP2405005B1 (en) | 2009-03-03 | 2010-02-16 | Microorganism which produces l-amino acid and method for producing l-amino acid using the same |
BRPI1013673-8A BRPI1013673B1 (pt) | 2009-03-03 | 2010-02-16 | Microorganismo que produz l-aminoácido e método para a produção de l-aminoácido usando o mesmo |
US13/254,072 US8623620B2 (en) | 2009-03-03 | 2010-02-16 | Microorganism which produces L-amino acid and method for producing L-amino acid using the same |
CN201080014196.XA CN102365363B (zh) | 2009-03-03 | 2010-02-16 | 产生l-氨基酸的微生物和利用其生产l-氨基酸的方法 |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020090018127A KR101058893B1 (ko) | 2009-03-03 | 2009-03-03 | L-아미노산 생산 미생물 및 이를 이용한 l-아미노산 생산방법 |
KR10-2009-0018127 | 2009-03-03 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2010101359A2 true WO2010101359A2 (ko) | 2010-09-10 |
WO2010101359A3 WO2010101359A3 (ko) | 2010-12-09 |
Family
ID=42710079
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/KR2010/000932 WO2010101359A2 (ko) | 2009-03-03 | 2010-02-16 | L-아미노산 생산 미생물 및 이를 이용한 l-아미노산 생산 방법 |
Country Status (6)
Country | Link |
---|---|
US (1) | US8623620B2 (ko) |
EP (1) | EP2405005B1 (ko) |
KR (1) | KR101058893B1 (ko) |
CN (1) | CN102365363B (ko) |
BR (1) | BRPI1013673B1 (ko) |
WO (1) | WO2010101359A2 (ko) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8129170B1 (en) | 2010-12-06 | 2012-03-06 | E.I. Du Pont De Nemours And Company | Recombinant bacteria having the ability to metabolize sucrose |
US8222000B2 (en) | 2010-12-06 | 2012-07-17 | E I Du Pont De Nemours And Company | Recombinant bacteria having the ability to metabolize sucrose |
WO2012099396A3 (en) * | 2011-01-18 | 2012-12-06 | Cj Cheiljedang Corporation | A microorganism having enhanced l-amino acids productivity and process for producing l-amino acids using the same |
US8686114B2 (en) | 2012-03-05 | 2014-04-01 | E I Du Pont De Nemours And Company | Variant sucrose transporter polypeptides |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR101327093B1 (ko) | 2012-01-06 | 2013-11-07 | 씨제이제일제당 (주) | L-아미노산을 생산할 수 있는 미생물 및 이를 이용하여 l-아미노산을 생산하는 방법 |
KR20140102393A (ko) * | 2013-02-13 | 2014-08-22 | 씨제이제일제당 (주) | L-쓰레오닌 생산능을 가지는 재조합 에스케리키아 속 미생물 및 이를 이용한 l-쓰레오닌의 생산방법 |
WO2015197082A1 (en) * | 2014-06-27 | 2015-12-30 | Glycom A/S | Oligosaccharide production |
US11926858B2 (en) | 2014-06-27 | 2024-03-12 | Glycom A/S | Oligosaccharide production |
EP3374491A4 (en) * | 2015-11-09 | 2019-07-03 | Cathay R&D Center Co., Ltd. | MODIFIED MEMBRANE PERMEABILITY |
KR101991206B1 (ko) * | 2018-11-29 | 2019-06-19 | 씨제이제일제당 (주) | cAMP 수용 단백질 변이체 및 이를 이용한 L-아미노산 제조방법 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20060084446A (ko) * | 2004-01-30 | 2006-07-24 | 아지노모토 가부시키가이샤 | L-아미노산 생산 미생물 및 l-아미노산 생산 방법 |
US7179623B2 (en) * | 2000-04-26 | 2007-02-20 | Ajinomoto Co., Inc. | Method of producing amino acids using E. coli transformed with sucrose PTS genes |
KR20080059604A (ko) * | 2005-09-27 | 2008-06-30 | 아지노모토 가부시키가이샤 | L-아미노산 생산 세균 및 l-아미노산 생산 방법 |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6610836B1 (en) * | 1999-01-29 | 2003-08-26 | Genome Therapeutics Corporation | Nucleic acid amino acid sequences relating to Klebsiella pneumoniae for diagnostics and therapeutics |
KR100576342B1 (ko) * | 2004-02-05 | 2006-05-03 | 씨제이 주식회사 | galR 유전자가 불활성화된 L-쓰레오닌 생성 미생물,그를 제조하는 방법 및 상기 미생물을 이용한L-쓰레오닌의 제조방법 |
JP2007117082A (ja) | 2005-09-27 | 2007-05-17 | Ajinomoto Co Inc | L−アミノ酸生産菌及びl−アミノ酸の製造法 |
-
2009
- 2009-03-03 KR KR1020090018127A patent/KR101058893B1/ko active IP Right Grant
-
2010
- 2010-02-16 EP EP10748903.1A patent/EP2405005B1/en active Active
- 2010-02-16 BR BRPI1013673-8A patent/BRPI1013673B1/pt active IP Right Grant
- 2010-02-16 CN CN201080014196.XA patent/CN102365363B/zh active Active
- 2010-02-16 WO PCT/KR2010/000932 patent/WO2010101359A2/ko active Application Filing
- 2010-02-16 US US13/254,072 patent/US8623620B2/en active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7179623B2 (en) * | 2000-04-26 | 2007-02-20 | Ajinomoto Co., Inc. | Method of producing amino acids using E. coli transformed with sucrose PTS genes |
KR20060084446A (ko) * | 2004-01-30 | 2006-07-24 | 아지노모토 가부시키가이샤 | L-아미노산 생산 미생물 및 l-아미노산 생산 방법 |
KR20080059604A (ko) * | 2005-09-27 | 2008-06-30 | 아지노모토 가부시키가이샤 | L-아미노산 생산 세균 및 l-아미노산 생산 방법 |
Non-Patent Citations (3)
Title |
---|
KOICHI HIRATSUKA ET AL.: 'Regulation of Sucrose-6-Phosphate Hydrolase Activity in Streptococcus mutans: Characterization of the scrR Gene' INFECTION AND IMMUNITY vol. 66, no. 8, 1998, pages 3736 - 3743, XP008151669 * |
See also references of EP2405005A2 * |
SHARON J. REID ET AL.: 'Sucrose utilisation in bacteria: genetic organisation and regulation' APPL MICROBIOL BIOTECHNOL. vol. 67, no. 3, 2005, pages 312 - 321, XP019331822 * |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8129170B1 (en) | 2010-12-06 | 2012-03-06 | E.I. Du Pont De Nemours And Company | Recombinant bacteria having the ability to metabolize sucrose |
US8222000B2 (en) | 2010-12-06 | 2012-07-17 | E I Du Pont De Nemours And Company | Recombinant bacteria having the ability to metabolize sucrose |
WO2012099396A3 (en) * | 2011-01-18 | 2012-12-06 | Cj Cheiljedang Corporation | A microorganism having enhanced l-amino acids productivity and process for producing l-amino acids using the same |
KR101261147B1 (ko) | 2011-01-18 | 2013-05-06 | 씨제이제일제당 (주) | L-아미노산의 생산능이 향상된 미생물 및 이를 이용하여 l-아미노산을 생산하는 방법 |
CN103443267A (zh) * | 2011-01-18 | 2013-12-11 | Cj第一制糖株式会社 | L-氨基酸生产力增强的微生物及利用其产生l-氨基酸的方法 |
US8835154B2 (en) | 2011-01-18 | 2014-09-16 | Cj Cheiljedang Corporation | Microorganism having enhanced L-amino acids productivity and process for producing L-amino acids using the same |
RU2549689C2 (ru) * | 2011-01-18 | 2015-04-27 | СиДжей ЧеилДжеданг Корпорейшн | Микроорганизм с повышенной продукцией l-аминокислот и способ получения l-аминокислот с его применением |
CN103443267B (zh) * | 2011-01-18 | 2015-11-25 | Cj第一制糖株式会社 | L-氨基酸生产力增强的微生物及利用其产生l-氨基酸的方法 |
US8686114B2 (en) | 2012-03-05 | 2014-04-01 | E I Du Pont De Nemours And Company | Variant sucrose transporter polypeptides |
Also Published As
Publication number | Publication date |
---|---|
US20120122163A1 (en) | 2012-05-17 |
EP2405005A9 (en) | 2014-10-22 |
BRPI1013673A2 (pt) | 2015-08-25 |
CN102365363A (zh) | 2012-02-29 |
KR101058893B1 (ko) | 2011-08-23 |
EP2405005A4 (en) | 2014-03-05 |
US8623620B2 (en) | 2014-01-07 |
WO2010101359A3 (ko) | 2010-12-09 |
KR20100099571A (ko) | 2010-09-13 |
EP2405005A2 (en) | 2012-01-11 |
BRPI1013673B1 (pt) | 2024-03-05 |
EP2405005B1 (en) | 2018-09-19 |
CN102365363B (zh) | 2014-05-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2010101359A2 (ko) | L-아미노산 생산 미생물 및 이를 이용한 l-아미노산 생산 방법 | |
WO2010101360A2 (ko) | L-아미노산 생산 미생물 및 이를 이용한 l-아미노산 생산 방법 | |
WO2010093182A2 (ko) | L-아미노산 생산용 미생물 및 이를 이용하여 l-아미노산을 생산하는 방법 | |
US7179623B2 (en) | Method of producing amino acids using E. coli transformed with sucrose PTS genes | |
RU2549689C2 (ru) | Микроорганизм с повышенной продукцией l-аминокислот и способ получения l-аминокислот с его применением | |
US9845513B2 (en) | Method of producing succinic acid and other chemicals using sucrose-containing feedstock | |
WO2012030130A2 (ko) | 수크로오즈와 글리세롤을 동시에 이용하는 신규 숙신산 생성 변이 미생물 및 이를 이용한 숙신산 제조방법 | |
KR101145943B1 (ko) | L-아미노산 생산능을 갖는 미생물 및 이를 이용하여 l-아미노산을 생산하는 방법 | |
WO2016182322A1 (ko) | L-트립토판 생산능을 갖는 에스케리키아속 미생물 및 이를 이용한 l-트립토판의 제조 방법 | |
WO2014126384A1 (ko) | L-쓰레오닌 생산능을 가지는 재조합 에스케리키아 속 미생물 및 이를 이용한 l-쓰레오닌의 생산방법 | |
WO2015122569A1 (ko) | L-쓰레오닌 생산능을 가지는 재조합 에스케리키아 속 미생물 및 이를 이용한 l-쓰레오닌의 생산방법 | |
EP3119875A1 (en) | Microorganisms producing l-amino acids and process for producing l-amino acids using the same | |
WO2015199406A1 (ko) | L-트립토판 생산능을 갖는 에스케리키아속 미생물 및 이를 이용한 l-트립토판의 제조 방법 | |
WO2015142021A1 (en) | Microorganisms having enhanced l-amino acids productivity and process for producing l-amino acids using the same | |
WO2016036209A1 (ko) | L-쓰레오닌 생산능이 향상된 미생물 및 이를 이용한 l-쓰레오닌 생산방법 | |
US9758772B2 (en) | L-threonine-producing microorganism and production method for L-threonine using the same |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
WWE | Wipo information: entry into national phase |
Ref document number: 201080014196.X Country of ref document: CN |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 10748903 Country of ref document: EP Kind code of ref document: A2 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2010748903 Country of ref document: EP |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 13254072 Country of ref document: US |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01A Ref document number: PI1013673 Country of ref document: BR |
|
ENP | Entry into the national phase |
Ref document number: PI1013673 Country of ref document: BR Kind code of ref document: A2 Effective date: 20110905 |