CN117964724A - Novel variant of transport protein and method for producing L-aromatic amino acid using same - Google Patents
Novel variant of transport protein and method for producing L-aromatic amino acid using same Download PDFInfo
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- CN117964724A CN117964724A CN202311729811.0A CN202311729811A CN117964724A CN 117964724 A CN117964724 A CN 117964724A CN 202311729811 A CN202311729811 A CN 202311729811A CN 117964724 A CN117964724 A CN 117964724A
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- amino acid
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- aromatic amino
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Classifications
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- 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
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
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- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
- C12P13/222—Phenylalanine
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- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
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- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
- C12P13/227—Tryptophan
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Abstract
The present invention relates to a novel variant of a transporter, which alters the activity of a protein by substituting one or more amino acids constituting the amino acid sequence of an ammonium transporter, an adenine transporter or an FMN/FAD transporter, so that a recombinant microorganism comprising the same can efficiently produce an L-aromatic amino acid, and a method for producing an L-aromatic amino acid using the same.
Description
The present invention is a divisional application filed for the application of 2023, 02, 14, 202380008895.0 and entitled "novel variant of transport protein and method for producing L-aromatic amino acid by using the same".
Technical Field
The present invention relates to novel variants of transport proteins and methods for producing L-aromatic amino acids using the same.
Background
Amino acids are classified into hydrophobic, hydrophilic, basic and acidic amino acids according to the nature of side chains, and amino acids having benzene rings therein are called aromatic amino acids. Among the aromatic amino acids, phenylalanine, tyrosine and tryptophan are essential amino acids that cannot be synthesized in living bodies, and belong to the high value-added industry that forms a market of $3000 million each year worldwide.
The production of aromatic amino acids can be carried out using wild-type strains obtained in a natural state or variant strains modified in such a manner as to enhance the amino acid productivity. In recent years, in order to improve the production efficiency of aromatic amino acids, genetic recombination techniques have been applied to microorganisms such as E.coli and coryneform bacteria which are used in many cases for producing useful substances such as L-amino acids, and various recombinant strains or variants having excellent L-aromatic amino acid productivity have been developed, and a method for producing L-aromatic amino acids using the same. In particular, the following attempts were made: the production of the corresponding amino acid is increased by targeting genes such as enzymes, transcription factors, transport proteins, etc. involved in the biosynthesis pathway of the L-aromatic amino acid, or inducing mutation in promoters regulating their expression. However, since the types of proteins such as enzymes, transcription factors, transport proteins, etc., which are directly or indirectly involved in the production of L-aromatic amino acids, are several tens of kinds, a great deal of research is actually required as to whether the L-aromatic amino acid production capacity is increased or not according to the activity change of such proteins.
Prior art literature
Patent literature
Korean patent No. 10-1830002
Disclosure of Invention
The object of the present invention is to provide novel transport protein variants.
In addition, the present invention provides polynucleotides encoding the above variants.
In addition, the present invention provides a transformant comprising the above variant or polynucleotide.
The present invention also provides a method for producing an L-aromatic amino acid using the transformant.
One embodiment of the present invention provides a variant selected from the following variants: an ammonium transporter variant consisting of the amino acid sequence of SEQ ID NO. 1, wherein glycine 363 of the amino acid sequence of SEQ ID NO. 3 is replaced with aspartic acid; an adenine transporter variant consisting of the amino acid sequence of SEQ ID NO. 5, wherein tryptophan No. 136 in the amino acid sequence of SEQ ID NO. 7 is replaced with a stop codon; and FMN/FAD transporter variants consisting of the amino acid sequence of SEQ ID NO. 9, wherein glycine 272 of the amino acid sequence of SEQ ID NO. 11 is replaced with glutamine.
As used herein, a "transporter" is a generic term for proteins responsible for moving various substances such as ions, chemicals, and proteins through a cell membrane, and is specific to the transported substance. Such transporter proteins are broadly classified into channel proteins that form a movement channel penetrating through inner and outer pores in a membrane to become a substance, and transporter proteins that move a binding site of a transported substance across the membrane by structural deformation of the protein.
As used in the present invention, "ammonium transporter", "adenine transporter" and "FMN/FAD transporter" are equivalent to transporter proteins, and have the property of specifically discharging only ammonium, adenine and FMN (flavin mononucleotide (flavin mononucleotide))/FAD (flavin adenine dinucleotide (FLAVIN ADENINE dinucleotide)), respectively.
The transporter may be a gene encoding each transporter or a sequence having substantial identity thereto. As used herein, "substantial identity" refers to a sequence homology of 70% or more, 80% or more, 90% or more, or 98% or more between any other nucleotide sequence and each gene sequence, i.e., the base sequence or nucleotide sequence, when the nucleotide sequence and any other nucleotide sequence are aligned in a maximally corresponding manner for analysis.
The ammonium transport protein is encoded by using an amtB gene and comprises the amino acid sequence of SEQ ID NO. 3.
According to one embodiment of the present invention, the amino acid sequence of SEQ ID NO. 3 may be derived from wild-type E.coli.
The adenine transporter protein is encoded by yicO genes and comprises the amino acid sequence of SEQ ID NO. 7.
According to one embodiment of the present invention, the amino acid sequence of SEQ ID NO. 7 may be derived from wild-type E.coli.
The FMN/FAD transport protein is encoded by yeeO genes and comprises the amino acid sequence of SEQ ID NO. 11.
According to one embodiment of the present invention, the amino acid sequence of SEQ ID NO. 11 may be derived from wild-type E.coli.
As used herein, a "variant" refers to a polypeptide in which one or more amino acids at the N-terminal, C-terminal and/or within the amino acid sequence of a particular gene are conservatively substituted (conservative substitution) and/or altered (modification) to differ from the amino acid sequence prior to mutation of the variant, but maintains functions or properties (properties). As used herein, "conservative substitutions" refer to the substitution of one amino acid for another that is structurally and/or chemically similar, with little or no effect on the activity of the protein or polypeptide. In addition, variants include variants with more than one N-terminal leader sequence or a portion of the transmembrane domain (transmembrane domain) deleted, or variants with a portion of the N-and/or C-terminal of the mature protein (protein). Such variants may have increased or unchanged or decreased ability compared to the polypeptide prior to mutation. In the present invention, variants may be used in combination with variants, alterations, variant polypeptides, variant proteins, variants, and the like.
The variant of the present invention is an ammonium transporter variant, which is an amino acid 363 in the amino acid sequence of SEQ ID NO. 3, i.e., glycine is substituted for aspartic acid, and may consist of the amino acid sequence of SEQ ID NO. 1.
The variant of the present invention is an adenine transporter variant in which tryptophan, which is the 136 th amino acid in the amino acid sequence of SEQ ID NO. 7, is replaced with a stop codon, and may be composed of the amino acid sequence of SEQ ID NO. 5.
The variant of the present invention is a FMN/FAD transporter variant in which glycine, which is the 272 th amino acid in the amino acid sequence of SEQ ID NO. 11, is replaced with glutamine, and may be composed of the amino acid sequence of SEQ ID NO. 9.
Another aspect of the invention provides polynucleotides encoding the ammonium transporter variants, adenine transporter variants, or FMN/FAD transporter variants described above.
The "polynucleotide (polynucleotide)" used in the present invention is a polymer (polymer) of nucleotides in which nucleotide monomers (monomers) are linked in a chain form by covalent bonds, and is a DNA or RNA strand of a predetermined length or more, more specifically, a polynucleotide fragment encoding the variant.
The above polynucleotide may comprise a base sequence encoding the amino acid sequence of SEQ ID NO. 1, 5 or 9.
According to one embodiment of the present invention, the polynucleotide may comprise a base sequence represented by SEQ ID NO. 2, 6 or 10.
More specifically, the polynucleotide encoding the variant of the ammonium transporter includes a base sequence represented by SEQ ID NO. 2, the polynucleotide encoding the variant of the adenine transporter includes a base sequence represented by SEQ ID NO. 6, and the polynucleotide encoding the variant of the FMN/FAD transporter includes a base sequence represented by SEQ ID NO. 10.
Another aspect of the invention provides a vector comprising a polynucleotide encoding the ammonium transporter variant, adenine transporter variant, or FMN/FAD transporter variant described above.
In addition, another aspect of the present invention provides a transformant comprising the above ammonium transporter variant, adenine transporter variant or FMN/FAD transporter variant, or a polynucleotide thereof.
The term "vector" as used herein refers to any type of nucleic acid sequence transport structure used as a means for delivering and expressing a target gene into a host cell. Unless otherwise indicated, such vectors may refer to the insertion of a supported nucleic acid sequence into a host cell gene for expression and/or expression separately. Such vectors include the necessary regulatory elements operably linked for expression of the gene insert, "operably linked (operably linked)" means that the gene of interest and its regulatory sequences are functionally bound to each other and linked in a manner that enables gene expression, "regulatory elements" include promoters for carrying out transcription, any operator sequences for regulating transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences that regulate termination of transcription and translation.
The vector used in the present invention is not particularly limited as long as it can replicate in a host cell, and any vector known in the art can be used. Examples of the vector include plasmids, cosmids, viruses, and phages in their natural or recombinant state. For example, as phage vectors or cosmid vectors, we15, M13, λmbl3, λmbl4, λ IXII, λ ASHII, λapii, λt10, λt11, charon4A, charon a, etc., and as plasmid vectors, pBR system, pUC system, pbluescript ii system, pGEM system, pTZ system, pCL system, pET system, etc., are available, but not limited thereto.
The above vectors may be representatively constructed as vectors for cloning or vectors for expression. The vector for expression may use a conventional vector for expressing a foreign gene or protein in a plant, animal or microorganism in the art, and may be constructed by various methods well known in the art.
The recombinant vector can be constructed by taking a prokaryotic cell or a eukaryotic cell as a host. For example, when the vector used is an expression vector and prokaryotic cells are used as a host, a strong promoter (e.g., plλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter) that allows transcription to proceed generally contains a ribosome binding site for translation initiation and transcription/translation termination sequences. When eukaryotic cells are used as hosts, the replication origins to be initiated in eukaryotic cells contained in the vector include, but are not limited to, f1 replication origins, SV40 replication origins, pMB1 replication origins, adenovirus replication origins, AAV replication origins, BBV replication origins, and the like. In addition, promoters derived from mammalian cell genomes (e.g., metallothionein promoters) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, tk promoter of HSV) may be utilized, and typically have polyadenylation sequences as transcription termination sequences.
The recombinant vector may include a selection marker for selecting transformants (host cells) transformed with the vector, and only cells expressing the selection marker may survive in the medium treated with the selection marker, thereby enabling selection of transformed cells. The selection markers include, but are not limited to, kanamycin, streptomycin, chloramphenicol, and the like.
By inserting the recombinant vector into a host cell, a transformant can be produced, which can be obtained by introducing the recombinant vector into an appropriate host cell. The host cell is a cell in which the above-described expression vector can be stably and continuously cloned or expressed, and any host cell known in the art can be used.
When a prokaryotic cell is transformed to produce a recombinant microorganism, a strain of Escherichia coli such as E.coli JM109、E.coli BL21、E.coli RR1、E.coli LE392、E.coli B、E.coli X 1776、E.coli W3110、E.coli XL1-Blue can be used as a host cell; bacillus strains such as bacillus subtilis and bacillus thuringiensis; various intestinal bacteria and strains such as Salmonella typhimurium, serratia marcescens and Pseudomonas species, etc., but are not limited thereto.
In the case of transforming eukaryotic cells for the production of recombinant microorganisms, yeasts (e.g., saccharomyces cerevisiae), insect cells, plant cells, and animal cells, for example, sp2/0, CHO K1, CHO DG44, PER.C6, W138, BHK, COS7, 293, hepG2, huh7, 3T3, RIN, MDCK cell lines, etc., can be used as host cells, but are not limited thereto.
The term "transformation" as used herein refers to a phenomenon in which a foreign DNA is introduced into a host cell to artificially cause a gene change, and the term "transformant (transformat)" refers to a host cell into which a foreign DNA is introduced and in which the expression of a target gene is stably maintained.
In the above transformation, an appropriate vector introduction technique is selected according to the host cell, so that the target gene or a recombinant vector comprising the same can be expressed in the host cell. For example, the vector introduction may be performed by electroporation (electroporation), thermal shock (heat-shock), calcium phosphate (CaPO 4) precipitation, calcium chloride (CaCl 2) precipitation, microinjection (microinjection), polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, lithium acetate-DMSO method, or a combination thereof, but is not limited thereto. The transformed gene may be included as long as it can be expressed in the host cell, and is not limited to insertion into the chromosome of the host cell or to being located extrachromosomally.
The above transformant includes a cell transfected, transformed or infected with the recombinant vector according to the present invention in an organism or in a test tube, and may be used as the same term as a recombinant host cell, a recombinant cell or a recombinant microorganism.
According to one embodiment of the present invention, the transformant may be a strain of Escherichia.
Examples of the strain of the genus Escherichia include Escherichia coli (ESCHERICHIA COLI), escherichia coli (ESCHERICHIA ALBERTII), escherichia blattae (ESCHERICHIA BLATTAE), escherichia fries (ESCHERICHIA FERGUSONII), escherichia hertz (ESCHERICHIA HERMANNII), and Escherichia wound (ESCHERICHIA VULNERIS), but are not limited thereto.
The transformant of the present invention may be a strain comprising the above-mentioned transporter variant or a polynucleotide encoding the same, or a strain comprising a vector containing the same; a strain expressing the transporter variant or polynucleotide; or a strain having an activity against the above-mentioned transporter variant, but is not limited thereto.
According to one embodiment of the present invention, the transformant may have L-aromatic amino acid productivity.
The transformant may have an L-aromatic amino acid-producing ability in nature or may be artificially provided with an L-aromatic amino acid-producing ability.
According to one embodiment of the present invention, the above transformant can be improved in L-aromatic amino acid productivity because the activity of an ammonium transporter variant, an adenine transporter variant or an FMN/FAD transporter is altered.
As used herein, "increased productivity" means that the productivity of L-aromatic amino acids is increased as compared to the parent strain. The parent strain refers to a wild-type strain or a mutant strain to be mutated, and includes a subject to be mutated directly or a subject to be transformed by a recombinant vector or the like. In the present invention, the parent strain may be a wild-type escherichia strain or an escherichia strain mutated from a wild-type strain.
The transformant according to the present invention shows an increased L-aromatic amino acid productivity as compared to the parent strain, particularly, an increase of more than 10% in L-aromatic amino acid productivity as compared to the parent strain, particularly, an increase of 10 to 80% (preferably 15 to 60%) by introducing an ammonium transporter variant, an adenine transporter variant or an FMN/FAD transporter variant, whereby the activity of each transporter is changed, and 3.5 to 20g of L-aromatic amino acid can be produced per 1 liter of strain culture broth.
Another aspect of the present invention provides a method for producing an L-aromatic amino acid, comprising the steps of: a step of culturing the transformant in a medium; and recovering the L-aromatic amino acid from the transformant or a medium in which the transformant is cultured.
The above-mentioned culture may be carried out according to a suitable medium and culture conditions known in the art, and the medium and culture conditions may be easily adjusted and used by those skilled in the art. Specifically, the medium may be a liquid medium, but is not limited thereto. The cultivation method may include, for example, batch cultivation (batch cultivation), continuous cultivation (continuous culture), fed-batch cultivation (fed-batch cultivation), or a combination thereof, but is not limited thereto.
According to one embodiment of the invention, the above-mentioned culture medium must meet the requirements of the particular strain in a suitable manner, and can be suitably deformed by a person skilled in the art. For the culture medium of the strain of Escherichia, reference may be made to well-known document (Manual of Methods for General Bacteriology.American Society for Bacteriology.Washington D.C.,USA,1981),, but is not limited thereto.
According to one embodiment of the present invention, the medium may contain various carbon sources, nitrogen sources and trace element components. As the carbon source which can be used, there are included sugars such as glucose, sucrose, lactose, fructose, maltose, starch, cellulose and the like and carbohydrates; oil and fat such as soybean oil, sunflower seed oil, castor seed oil, coconut oil and the like; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerin and ethanol; organic acids such as acetic acid. These substances may be used alone or in the form of a mixture, but are not limited thereto. As nitrogen sources that can be used, peptone, yeast extract, broth, malt extract, corn steep liquor, soybean meal and urea or inorganic compounds, for example, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate can be included. The nitrogen source may also be used alone or in the form of a mixture, but is not limited thereto. As the supply source of phosphorus that can be used, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or corresponding sodium-containing salts may be included, but are not limited thereto. The medium may contain a metal salt such as magnesium sulfate or iron sulfate required for growth, but is not limited thereto. In addition, essential growth substances such as amino acids and vitamins may be contained. In addition, precursors suitable for the culture medium may be used. The above-mentioned medium or individual components may be added to the culture broth in batches or continuously by an appropriate means during the culture, but are not limited thereto.
According to an embodiment of the present invention, during the cultivation, a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid may be added to the microorganism culture solution in an appropriate manner to adjust the pH of the culture solution. In addition, during the culture, an antifoaming agent such as fatty acid polyglycol ester may be used to suppress bubble generation. Further, in order to maintain the aerobic state of the culture, oxygen or an oxygen-containing gas (e.g., air) may be injected into the culture. The temperature of the culture solution may be generally 20℃to 45℃and, for example, may be 25℃to 40 ℃. The incubation time may be continued until the desired throughput of the useful substance is obtained, for example, may be 10 to 160 hours.
According to one embodiment of the present invention, in the step of recovering an L-aromatic amino acid from the above-mentioned cultured transformant or the medium in which the transformant is cultured, the produced L-aromatic amino acid may be collected or recovered from the medium according to a culture method and by using an appropriate method known in the art. For example, centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobicity, and size exclusion), and the like may be used, but are not limited thereto.
According to one embodiment of the present invention, in the step of recovering an L-aromatic amino acid, the medium may be centrifuged at a low speed to remove biomass, and the obtained supernatant may be separated by ion exchange chromatography.
According to one embodiment of the present invention, the step of recovering the L-aromatic amino acid may include a step of purifying the L-aromatic amino acid.
According to one embodiment of the present invention, the L-aromatic amino acid may be 1 or more selected from the group consisting of L-tryptophan, L-phenylalanine and L-tyrosine.
The transport protein variant according to the present invention alters protein activity by substituting one or more amino acids constituting the amino acid sequence of ammonium transport protein, adenine transport protein or FMN/FAD transport protein, so that a recombinant microorganism comprising the same can efficiently produce L-aromatic amino acids.
Drawings
FIG. 1 shows the structure of a plasmid pDSG according to an embodiment of the present invention.
FIG. 2 shows the structure of plasmid pDS9 according to an embodiment of the present invention.
Detailed Description
The present invention will be described in more detail below. However, the description is merely illustrative for the purpose of facilitating understanding of the present invention, and the scope of the present invention is not limited to such illustrative description.
Example 1 preparation of strains expressing variants of ammonium Transporter
In order to confirm the effect of the variant in which glycine 363 is substituted with aspartic acid in the amino acid sequence of ammonium transporter (SEQ ID NO: 3) on the production of L-aromatic amino acids, vectors expressing the above ammonium transporter variants and strains into which the above vectors were introduced were made. For gene insertion of the intra-strain ammonium transporter variant, plasmids pDSG and pDS9 were used and made as follows.
Here, plasmid pDSG has an origin of replication that functions only in E.coli, has an ampicillin resistance gene and a leader RNA (gRNA) expression mechanism. Plasmid pDS9 has an origin of replication which functions only in E.coli, with kanamycin resistance gene, the λRed gene (exo, bet and gam) and the CAS9 expression mechanism derived from Streptococcus pyogenes (Streptococcus pyogenes).
1-1 Preparation of vector pDSG-amtB for transformation (Gly 363 Asp)
By performing PCR using the gDNA of E.coli (ESCHERICHIA COLI) MG1655 (KCTC 14419 BP) as a template and using the primer pair of the primer 7 and the primer 9 and the primer pair of the primer 8 and the primer 10, respectively, an upstream (upstream) fragment of the No. 363 amino acid variation of the E.coli ambB gene encoding an ammonium transporter was obtained, and a downstream (down) fragment of the No. 363 amino acid variation of the E.coli ambB gene was obtained using the primer pair of the primer 11 and the primer 13 and the primer pair of the primer 12 and the primer 14. In this case, each of the upstream (upstream) and downstream (downstream) fragments includes a sequence in which glycine (Gly) which is amino acid residue 363 of the ambb gene is changed to aspartic acid (Asp). Here, the use of TAKARA PRIMESTAR Max DNApolymerase, PCR amplification conditions by the polymerase was performed by repeating denaturation at 95℃for 10 seconds, annealing at 57℃for 15 seconds, and polymerization at 72℃for 10 seconds 30 times.
4 PDSG gene fragments were obtained by performing PCR using the plasmid pDSG as a template and the primer pairs of the primers 3 and 5, the primer pair of the primers 4 and 6, the primer pair of the primers 15 and 1, and the primer pair of the primers 16 and 2, respectively. In this case, each gene fragment was made to contain a gRNA sequence targeting Gly 363 of the amtB gene. The gRNA selects the NGG pre 20mer of the sequence to be mutated. Here, the use of TAKARA PRIMESTAR Max DNApolymerase, PCR amplification conditions by the polymerase was performed by repeating denaturation at 95℃for 10 seconds, annealing at 57℃for 15 seconds and polymerization at 72℃for 15 seconds 30 times.
Using self-assembled cloning (BioTechniques 51:55-56 (July 2011)), the obtained amino acids 363 of the amtB gene were cloned upstream (upstream) and downstream (downstream), and 4 pDSG gene fragments, thereby obtaining a recombinant plasmid designated pDSG-amtB (Gly 363 Asp).
1-2 Preparation of L-tryptophan or L-phenylalanine producing Strain into which ammonium Transporter variant amtB (Gly 363 Asp) was introduced
Coli KCCM13013P and KCCM10016 were used as parent strains for the production of L-tryptophan and L-phenylalanine producing strains.
Plasmid pDS9 was transformed into KCCM13013P strain or KCCM10016 strain 1 time, and after culturing in LB-Km (containing 25g/L LB liquid medium and 50mg/L kanamycin) solid medium, colonies with kanamycin resistance were selected. The pDSG-amtB (Gly 363 Asp) plasmid was transformed 2 times into selected colonies, cultured in LB-Amp & Km (containing 25g/L LB liquid medium, 100mg/L ampicillin and 50mg/L kanamycin) solid medium, and after ampicillin and kanamycin resistant colonies were selected, PCR was performed using primer pair of primer 17 and primer 18, thereby obtaining gene fragments. The use of TAKARA PRIMESTAR Max DNApolymerase, PCR amplification conditions for the polymerase was performed by repeating denaturation at 95℃for 10 seconds, annealing at 57℃for 10 seconds, and polymerization at 72℃for 15 seconds 30 times. The order Macrogen confirms the sequence of the gene fragment obtained with the primer pair of primer 17 and primer 18.
The selected 2 transformants were subcultured 7 times in LB liquid medium, and colonies were selected in LB solid medium. Each colony was selectively cultured in LB, LB-Amp and LB-Km solid medium, respectively. Colonies that grew in LB solid medium and did not grow in LB-Amp and LB-Km solid medium were selected. The strains produced by the method described above were designated KCCM13013P_ pDSG-amtB (Gly 363 Asp) and KCCM10016_ pDSG-amtB (Gly 363 Asp), respectively.
The primer sequences used in example 1 are shown in Table 1 below.
TABLE 1
Experimental example 1 evaluation of L-aromatic amino acid production ability of variant introduced with ammonium transport protein variant
The L-tryptophan or L-phenylalanine production capacity of the parent strain (KCCM 13013P and KCCM 10016) and the variant strain (KCCM 13013P_amtB (Gly 363 Asp) and KCCM10016_amtB (Gly 363 Asp)) into which the ammonium transporter variant was introduced were compared.
Each strain (parent strain or variant strain) was inoculated by 1% by volume and shake-cultured at 37℃for 72 hours under conditions of 200rpm in a 100mL flask containing 10mL of the tryptophan-producing medium or the phenylalanine-producing medium of Table 2 below. After the completion of the culture, the concentration of L-tryptophan or L-phenylalanine in the medium was measured by HPLC (Agilent), and the results are shown in tables 3 and 4 below, respectively.
TABLE 2
TABLE 3
TABLE 44
As shown in tables 3 and 4 above, it was confirmed that the variant strains KCCM13013P_amtB (Gly 363 Asp) and KCCM10016_amtB (Gly 363 Asp) into which the ammonium transporter variants were introduced had an increase in L-tryptophan and L-phenylalanine production by about 19% and 33%, respectively, as compared with the parent strains KCCM13013P and KCCM 10016.
EXAMPLE 2 production of strains expressing variant adenine transporter
In order to confirm the effect of the variant in which tryptophan at position 136 was replaced with a stop codon in the amino acid sequence of adenine transporter (SEQ ID NO: 7) on the production of L-aromatic amino acids, a vector expressing the variant of adenine transporter and a strain into which the vector was introduced were produced. For gene insertion of the adenine transporter variant in the strain, the plasmid pDSG and pDS9 of example 1 were used and made as follows.
2-1 Preparation of transformation vector pDSG-yicO (Trp 136 Stop)
By performing PCR using the gDNA of Escherichia coli (ESCHERICHIA COLI) MG1655 (KCTC 14419 BP) as a template and using the primer set of the primer 7 and the primer 9 and the primer set of the primer 8 and the primer 10, respectively, an upstream (upstream) fragment of the 136 th amino acid variation of the Escherichia coli yicO gene encoding adenine transporter was obtained, and a downstream (downstream) fragment of the 136 th amino acid variation of the Escherichia coli yicO gene was obtained using the primer set of the primer 11 and the primer 13 and the primer set of the primer 12 and the primer 14. In this case, the upstream (upstream) and downstream (downstream) fragments were each made to include a sequence in which tryptophan (Trp) as amino acid residue 136 of yicO gene was changed to a Stop codon (Stop). Here, the use of TAKARA PRIMESTAR Max DNApolymerase, PCR amplification conditions by the polymerase was performed by repeating denaturation at 95℃for 10 seconds, annealing at 57℃for 15 seconds, and polymerization at 72℃for 10 seconds 30 times.
4 PDSG gene fragments were obtained by performing PCR using the plasmid pDSG as a template and the primer pairs of the primers 3 and 5, the primer pair of the primers 4 and 6, the primer pair of the primers 15 and 1, and the primer pair of the primers 16 and 2, respectively. In this case, each gene fragment was made to contain a gRNA sequence targeting Trp No. 136 of yicO gene. The gRNA selects the NGG pre 20mer of the sequence to be mutated. Here, the use of TAKARA PRIMESTAR Max DNApolymerase, PCR amplification conditions by the polymerase was performed by repeating denaturation at 95℃for 10 seconds, annealing at 57℃for 15 seconds and polymerization at 72℃for 15 seconds 30 times.
The obtained upstream (upstream) and downstream (down stream) of amino acid 136 of yicO gene and 4 pDSG gene fragments were cloned by self-assembled cloning (BioTechniques 51:55-56 (July 2011)), thereby obtaining a recombinant plasmid designated as pDSG-yicO (Trp 136 Stop).
2-2 Production of L-tryptophan or L-phenylalanine producing Strain into which adenine transporter variant yicO (Trp 136 Stop) was introduced
Coli KCCM13013P and KCCM10016 were used as parent strains for the production of L-tryptophan and L-phenylalanine producing strains.
Plasmid pDS9 was transformed into KCCM13013P strain or KCCM10016 strain 1 time, and after culturing in LB-Km (containing 25g/L LB liquid medium and 50mg/L kanamycin) solid medium, colonies with kanamycin resistance were selected. The pDSG-yicO (Trp 136 Stop) plasmid was transformed into a selected colony 2 times, cultured in LB-Amp & Km (containing 25g/L LB liquid medium, 100mg/L ampicillin and 50mg/L kanamycin) solid medium, and after ampicillin and kanamycin resistant colonies were selected, PCR was performed using primer pair of primer 17 and primer 18 to obtain a gene fragment. The use of TAKARA PRIMESTAR Max DNApolymerase, PCR amplification conditions for the polymerase was performed by repeating denaturation at 95℃for 10 seconds, annealing at 57℃for 10 seconds, and polymerization at 72℃for 15 seconds 30 times. The order Macrogen confirms the sequence of the gene fragment obtained with the primer pair of primer 17 and primer 18.
The selected 2 transformants were subcultured 7 times in LB liquid medium, and colonies were selected in LB solid medium. Each colony was selectively cultured in LB, LB-Amp and LB-Km solid medium, respectively. Colonies that grew in LB solid medium and did not grow in LB-Amp and LB-Km solid medium were selected. The strains produced by the method described above were named KCCM13013p_ yicO (Trp 136 Stop) and KCCM10016_ yicO (Trp 136 Stop), respectively.
The primer sequences used in example 2 are shown in Table 5 below.
TABLE 5
Experimental example 2 evaluation of L-aromatic amino acid production ability of variant introduced with adenine transporter variant
The L-tryptophan or L-phenylalanine production capacity of the parent strain (KCCM 13013P and KCCM 10016) and the variant strain (KCCM 13013P_ yicO (Trp 136 Stop) and KCCM10016_ yicO (Trp 136 Stop)) into which the adenine transporter variant was introduced were compared. The concentrations of L-tryptophan and L-phenylalanine were measured in the same manner as in Experimental example 1, and the results are shown in tables 6 and 7, respectively, below.
TABLE 6
TABLE 7
As shown in tables 6 and 7 above, it was confirmed that the variant strains KCCM13013P_ yicO (Trp 136 Stop) and KCCM10016_ yicO (Trp 136 Stop) into which adenine transporter variants were introduced had about 26% and 19% increased L-tryptophan and L-phenylalanine production, respectively, as compared with the parent strains KCCM13013P and KCCM 10016.
EXAMPLE 3 preparation of strains expressing FMN/FAD transporter variants
In order to confirm the effect of a variant in which glycine at position 272 in the amino acid sequence of FMN/FAD transporter (SEQ ID NO: 11) was replaced with glutamine on the production of L-aromatic amino acids, vectors expressing the above FMN/FAD transporter variants and strains into which the above vectors were introduced were prepared. For gene insertion of the in-strain FMN/FAD transporter variant, plasmid pDSG and pDS9 were used by the same method as in example 1 and were made as follows.
3-1 Production of vector pDSG-yeeO for transformation (Gly 272 Glu)
By performing PCR using E.coli (ESCHERICHIA COLI) MG1655 (KCTC 14419 BP) gDNA as a template and using the primer set of the primer 7 and the primer 9 and the primer set of the primer 8 and the primer 10, respectively, an upstream (upstream) fragment of the 272 th amino acid variation of the E.coli yeeO gene encoding FMN/FAD transporter was obtained, and a downstream (downstream) fragment of the 272 th amino acid variation of the E.coli yeeO gene was obtained using the primer set of the primer 11 and the primer 13 and the primer set of the primer 12 and the primer 14. In this case, each of the upstream (upstream) and downstream (downstream) fragments includes a sequence in which glycine (Gly) which is the 272 th amino acid residue of yeeO gene is changed to glutamine (Glu). Here, the use of TAKARA PRIMESTAR Max DNApolymerase, PCR amplification conditions by the polymerase was performed by repeating denaturation at 95℃for 10 seconds, annealing at 57℃for 15 seconds, and polymerization at 72℃for 10 seconds 30 times.
4 PDSG gene fragments were obtained by performing PCR using the plasmid pDSG as a template and the primer pairs of the primers 3 and 5, the primer pair of the primers 4 and 6, the primer pair of the primers 15 and 1, and the primer pair of the primers 16 and 2, respectively. In this case, each gene fragment was made to contain a gRNA sequence targeting Gly No. 272 of yeeO gene. The gRNA selects the NGG pre 20mer of the sequence to be mutated. Here, the use of TAKARA PRIMESTAR Max DNApolymerase, PCR amplification conditions by the polymerase was performed by repeating denaturation at 95℃for 10 seconds, annealing at 57℃for 15 seconds and polymerization at 72℃for 15 seconds 30 times.
Using self-assembled cloning (BioTechniques 51:55-56 (July 2011)), the obtained up-stream and down-stream (Down-stream) amino acid 272 of yeeO gene and 4 pDSG gene fragments were cloned to obtain a recombinant plasmid, which was named pDSG-yeeO (Gly 272 Glu).
3-2 Production of L-tryptophan or L-phenylalanine producing Strain into which FMN/FAD transporter variant yeeO (Gly 272 Glu) was introduced
Coli KCCM13013P and KCCM10016 were used as parent strains for the production of L-tryptophan and L-phenylalanine producing strains.
Plasmid pDS9 was transformed into KCCM13013P strain or KCCM10016 strain 1 time, and after culturing in LB-Km (containing 25g/L LB liquid medium and 50mg/L kanamycin) solid medium, colonies with kanamycin resistance were selected. The pDSG-yeeO (Gly 272 Glu) plasmid was transformed into a selected colony 2 times, cultured in LB-Amp & Km (containing 25g/L LB liquid medium, 100mg/L ampicillin and 50mg/L kanamycin) solid medium, and after ampicillin and kanamycin resistant colonies were selected, PCR was performed using the primer pair of primer 17 and primer 18 to obtain a gene fragment. The polymerase was used TAKARA PRIMESTAR Max DNA polymerase, and PCR amplification conditions were performed by repeating denaturation at 95℃for 10 seconds, annealing at 57℃for 10 seconds, and polymerization at 72℃for 15 seconds 30 times. The order Macrogen confirms the sequence of the gene fragment obtained with the primer pair of primer 17 and primer 18.
The selected 2 transformants were subcultured 7 times in LB liquid medium, and colonies were selected in LB solid medium. Each colony was selectively cultured in LB, LB-Amp and LB-Km solid medium, respectively. Colonies that grew in LB solid medium and did not grow in LB-Amp and LB-Km solid medium were selected. Strains produced by the method described above were designated as KCCM13013P_ yeeO (Gly 272 Glu) and KCCM10016_ yeeO (Gly 272 Glu), respectively.
The primer sequences used in example 3 are shown in Table 8 below.
TABLE 8
Experimental example 3 evaluation of L-aromatic amino acid production ability of variant introduced with FMN/FAD transporter variant
The L-tryptophan or L-phenylalanine production capacity of the parent strain (KCCM 13013P and KCCM 10016) and the variant strain (KCCM 13013P_ yeeO (Gly 272 Glu) and KCCM10016_ yeeO (Gly 272 Glu)) into which the FMN/FAD transporter variant was introduced were compared. The concentrations of L-tryptophan and L-phenylalanine were measured in the same manner as in Experimental example 1, and the results thereof are shown in tables 9 and 10, respectively, below.
TABLE 9
TABLE 10
As shown in tables 9 and 10 above, it was confirmed that the variant strains KCCM13013P_ yeeO (Gly 272 Glu) and KCCM10016_ yeeO (Gly 272 Glu) into which the FMN/FAD transporter variants were introduced had an increase in L-tryptophan and L-phenylalanine production by about 21% and 43%, respectively, as compared with the parent strains KCCM13013P and KCCM 10016.
The present invention has been studied so far about its preferred embodiments. Those skilled in the art to which the invention pertains will appreciate that the invention may be practiced in modified forms without deviating from the essential characteristics of the invention. Accordingly, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the invention is indicated in the claims rather than in the foregoing description and all differences within the scope equivalent thereto are intended to be included in the present invention.
[ Preservation information ]
1. Coli (ESCHERICHIA COLI) DS9H (MG 1655)
(1) Preservation unit: KCTC Korea Collection of typical cultures
(2) Address: korean full Roth Malay Kogyi Ipsin-gil 181, 56212 Korea institute of vital engineering (Korea Research Institute of Bioscience and Biotechnology, KRIBB)
(3) Preservation date: 2020, 12 months and 28 days
(4) Preservation number: KCTC 14419BP
(5) Classification naming of biological materials: coli (ESCHERICHIA COLI).
2. Coli (ESCHERICHIA COLI) W0G
(1) Preservation unit: KCCM Korean center for microorganism collection
(2) Address: hongji inner 2A road No. 45 academic circle building, post code 03641 in the Siemens gate district of Korea head city
(3) Preservation date: 2021, 6, 22
(4) Preservation number: KCCM13013P
(5) Classification naming of biological materials: coli (ESCHERICHIA COLI).
3. Coli (ESCHERICHIA COLI) MWOR 247
(1) Preservation unit: KCCM Korean center for microorganism collection
(2) Address: hongji-1-hole, 361-221, academic circle building, zip code 120-091 in the Siemens of Korea head city
(3) Preservation date: 10/24/1992
(4) Preservation number: KCCM-10016
(5) Classification naming of biological materials: coli (ESCHERICHIA COLI).
Claims (7)
1. An adenine transporter variant consisting of the amino acid sequence of SEQ ID No. 5, wherein tryptophan No. 136 in the amino acid sequence of SEQ ID No. 7 is replaced with a stop codon.
2. A polynucleotide encoding the variant of claim 1.
3. A transformant comprising the variant of claim 1 or the polynucleotide of claim 2.
4. A transformant according to claim 3, wherein the transformant is a strain of Escherichia.
5. The transformant according to claim 3, wherein the transformant has L-aromatic amino acid productivity.
6. A method for producing an L-aromatic amino acid, comprising the steps of:
a step of culturing the transformant according to claim 3 in a medium; and
Recovering the L-aromatic amino acid from the transformant or a medium in which the transformant is cultured.
7. The method according to claim 6, wherein the L-aromatic amino acid is 1 or more selected from the group consisting of L-tryptophan, L-phenylalanine and L-tyrosine.
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| KR1020220020012A KR20230123544A (en) | 2022-02-16 | 2022-02-16 | Novel variant of transporter and method for preparing L-aromatic amino acid using the same |
| CN202380008895.0A CN117043178A (en) | 2022-02-16 | 2023-02-14 | Novel variant of transport protein and method for producing L-aromatic amino acid using same |
| PCT/KR2023/002096 WO2023158175A1 (en) | 2022-02-16 | 2023-02-14 | Novel transport protein variant and method for producing l-aromatic amino acid using same |
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| CN202311729811.0A Pending CN117964724A (en) | 2022-02-16 | 2023-02-14 | Novel variant of transport protein and method for producing L-aromatic amino acid using same |
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