Patent Application
20240218405
The present disclosure relates to a Corynebacterium glutamicum variant with improved L-lysine producing ability and a method of producing L-lysine using the same.
L-Lysine is an essential amino acid that is not synthesized in the human or animal body and must be supplied from the outside, and is generally produced through fermentation using microorganisms such as bacteria or yeast. In L-lysine production, naturally obtained wild-type strains or variants modified to have enhanced L-lysine producing ability thereof may be used. Recently, in order to improve the production efficiency of L-lysine, various recombinant strains or variants with excellent L-lysine producing ability and methods of producing L-lysine using the same have been developed by applying a genetic recombination technology to microorganisms such as Escherichia coli and Corynebacterium, etc., which are widely used in the production of L-amino acids and other useful substances.
According to Korean Patent Nos. 10-0838038 and 10-2139806, nucleotide sequences of genes encoding proteins including enzymes related to L-lysine production or amino acid sequences thereof are modified to increase expression of the genes or to remove unnecessary genes and thereby improve the L-lysine producing ability. In addition, Korean Patent Publication No. 10-2020-0026881 discloses a method of replacing the existing promoter of a gene with a promoter with strong activity in order to increase expression of the gene encoding the enzyme involved in L-lysine production.
As described, a variety of methods to increase the L-lysine producing ability are being developed. Nevertheless, since there are dozens of types of proteins such as enzymes, transcription factors, transport proteins, etc. which are directly or indirectly involved in the L-lysine production, it is necessary to conduct many studies on whether or not the L-lysine producing ability is increased according to changes in the activities of these proteins.
An object of the present disclosure is to provide a Corynebacterium glutamicum variant with improved L-lysine producing ability.
Further, another object of the present disclosure is to provide a method of producing L-lysine using the variant.
The present inventors have studied to develop a novel variant with improved L-lysine producing ability using a Corynebacterium glutamicum strain, and as a result, they found that L-lysine production is increased by substituting a nucleotide sequence at a specific position in a promoter of aspB gene encoding aspartate aminotransferase, which is involved in the supply of a lysine precursor aspartate in the L-lysine biosynthetic pathway, thereby completing the present disclosure.
An aspect of the present disclosure provides a Corynebacterium glutamicum variant with improved L-lysine producing ability by enhancing the activity of aspartate aminotransferase.
As used herein, the term “aspartate aminotransferase (aspartate transaminase)” refers to an enzyme that catalyzes a conversion reaction of oxaloacetate to aspartate in the L-lysine biosynthetic pathway.
According to a specific embodiment of the present disclosure, the aspartate aminotransferase may be derived from a strain of the genus Corynebacterium. Specifically, the strain of the genus Corynebacterium may be Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium callunae, Corynebacterium suranareeae, Corynebacterium Corynebacterium lubricantis, Corynebacterium doosanense, efficiens, Corynebacterium uterequi, Corynebacterium stationis, Corynebacterium pacaense, Corynebacterium singulare, Corynebacterium humireducens, Corynebacterium marinum, Corynebacterium halotolerans, Corynebacterium spheniscorum, Corynebacterium freiburgense, Corynebacterium striatum, Corynebacterium canis, Corynebacterium ammoniagenes, Corynebacterium renale, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium caspium, Corynebacterium testudinoris, Corynebacaterium pseudopelargi, or Corynebacterium flavescens, but is not limited thereto.
As used herein, “enhancing the activity” means that expression levels of genes encoding proteins such as target enzymes, transcription factors, transport proteins, etc. are increased by newly introducing or enhancing the genes, as compared to those of a wild-type strain or a strain before modification. Such enhancement of the activity also includes the case where activity of the protein itself is increased through substitution, insertion, or deletion of the nucleotide encoding the gene, or a combination thereof, as compared to activity of the protein originally possessed by a microorganism, and the case where the overall enzyme activity in cells is higher than that of the wild-type strain or the strain before modification, due to increased expression or increased translation of the genes encoding the same, and a combination thereof.
According to a specific embodiment of the present disclosure, enhancement of the activity of aspartate aminotransferase may induce a site-specific mutation in a promoter of a gene encoding aspartate aminotransferase.
According to a specific embodiment of the present disclosure, the promoter of the gene encoding aspartate aminotransferase may be represented by a nucleotide sequence of SEQ ID NO: 1.
As used herein, the “promoter” refers to a specific region of DNA that regulates gene transcription by including the binding site for RNA polymerase that initiates mRNA transcription of a gene of interest, and is generally located upstream of the transcription start site. The promoter in prokaryotes is defined as a region near the transcription start site where RNA polymerase binds, and generally consists of two short nucleotide sequences at −10 and −35 base-pair regions upstream from the transcription start site. In the present disclosure, the promoter mutation is that the promoter is improved to have high activity, as compared to a wild-type promoter, and may increase the expression of genes located downstream by inducing mutations in the promoter region located upstream of the transcription start site.
According to a specific embodiment of the present disclosure, the enhancement of the activity of aspartate aminotransferase may be substitution of one or more bases at −100 to −10 regions upstream from the transcription start site in the promoter sequence of the gene encoding aspartate aminotransferase.
More specifically, the promoter mutation of the present disclosure may be consecutive or non-consecutive substitution of one or more bases at −100 to −10 regions, preferably, consecutive or non-consecutive substitution of one, two, three, four, or five bases at −95 to −15 regions, −95 to −55 regions, −55 to −40 regions, or −30 to −15 regions.
According to one exemplary embodiment of the present disclosure, C which is a nucleotide sequence at −22 region of the promoter sequence of aspB gene encoding aspartate aminotransferase of the Corynebacterium glutamicum strain is substituted with G to obtain a Corynebacterium glutamicum variant having a new promoter sequence of aspB gene. Such a Corynebacterium glutamicum variant may include the mutated promoter of aspB gene, which is represented by a nucleotide sequence of SEQ ID NO: 2.
Further, according to one exemplary embodiment of the present disclosure, T which is a nucleotide sequence at −45 region of the promoter sequence of aspB gene encoding aspartate aminotransferase of the Corynebacterium glutamicum strain is substituted with A to obtain a Corynebacterium glutamicum variant having a new promoter sequence of aspB gene. Such a Corynebacterium glutamicum variant may include the mutated promoter of aspB gene, which is represented by a nucleotide sequence of SEQ ID NO: 3.
According to one exemplary embodiment of the present disclosure, T which is a nucleotide sequence at −88 region of the promoter sequence of aspB gene encoding aspartate aminotransferase of the Corynebacterium glutamicum strain is substituted with A to obtain a Corynebacterium glutamicum variant having a new promoter sequence of aspB gene. Such a Corynebacterium glutamicum variant may include the mutated promoter of aspB gene, which is represented by a nucleotide sequence of SEQ ID NO: 4.
As used herein, the “improved production ability” means increased L-lysine productivity, as compared to that of a parent strain. The parent strain refers to a wild-type or variant strain that is a subject of mutation, and includes a subject that is directly mutated or transformed with a recombinant vector, etc. In the present disclosure, the parent strain may be a wild-type Corynebacterium glutamicum strain or a strain mutated from the wild-type.
According to a specific embodiment of the present disclosure, the parent strain is a variant in which mutations are induced in the sequences of genes (e.g., lysC, zwf, and hom genes) involved in the lysine production, and may be a Corynebacterium glutamicum strain (hereinafter referred to as ‘Corynebacterium glutamicum DS1 strain’) deposited at the Korean Culture Center of Microorganisms on Apr. 2, 2021, with Accession No. KCCM12969P.
The Corynebacterium glutamicum variant having the improved L-lysine producing ability of the present disclosure may include a mutated promoter sequence of the gene encoding aspartate aminotransferase.
According to a specific embodiment of the present disclosure, the variant may include any one of nucleotide sequences represented by SEQ ID NOS: 2 to 4 as the promoter sequence of the aspartate aminotransferase gene.
According to one exemplary embodiment of the present disclosure, the variant may include the promoter mutation of aspB gene encoding aspartate aminotransferase, thereby exhibiting the increased L-lysine producing ability, as compared to the parent strain, and in particular, may exhibit 3% or more, specifically 3% to 40%, and more specifically 5% to 30% increase in the L-lysine production, as compared to the parent strain, thereby producing 65 g to 90 g of L-lysine, preferably 70 g to 80 g of L-lysine per 1 L of the culture of the strain.
The Corynebacterium glutamicum variant according to a specific embodiment of the present disclosure may be achieved through a recombinant vector including a variant in which part of the promoter sequence of the gene encoding aspartate aminotransferase in the parent strain is substituted.
As used herein, the “part” means not all of a nucleotide sequence or polynucleotide sequence, and may be 1 to 300, preferably 1 to 100, and more preferably 1 to 50, but is not limited thereto.
As used herein, the “variant” refers to a promoter variant in which one or more bases at −100 to −10 regions in the promoter sequence of the aspartate aminotransferase gene involved in the L-lysine biosynthesis are substituted.
According to a specific embodiment of the present disclosure, variants, each in which the nucleotide sequence at −22, −45, or −88 region in the promoter sequence of the aspartate aminotransferase gene is substituted with G, A, or A, may have the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, respectively.
As used herein, the “vector” is an expression vector capable of expressing a target protein in a suitable host cell, and refers to a gene construct including essential regulatory elements which are operably linked so that a gene insert is expressed. Here, “operably linked” means that a gene requiring expression and a regulatory sequence thereof are functionally linked to each other to induce gene expression, and the “regulatory elements” include a promoter for performing transcription, any operator sequence for controlling the transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling termination of transcription and translation. Such a vector may include a plasmid vector, a cosmid vector, a bacteriophage vector, a viral vector, etc., but is not limited thereto.
As used herein, the “recombinant vector” is transformed into a suitable host cell and then replicated independently of the genome of the host cell, or may be integrated into the genome itself. In this regard, the “suitable host cell”, where the vector is replicable, may include the origin of replication which is a particular nucleotide sequence at which replication is initiated.
For the transformation, an appropriate technology of introducing the vector is selected depending on the host cell to express the target gene in the host cell. For example, introduction of the vector may be performed by electroporation, heat-shock, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2)) precipitation, microinjection, a polyethylene glycol (PEG) method, a DEAE-dextran method, a cationic liposome method, a lithium acetate-DMSO method, or combinations thereof. As long as the transformed gene may be expressed within the host cell, it may be included without limitation, regardless of whether or not the gene is inserted into the chromosome of the host cell or located outside the chromosome.
The host cells include cells transfected, transformed, or infected with the recombinant vector or polynucleotide of the present disclosure in vivo or in vitro. Host cells including the recombinant vector of the present disclosure are recombinant host cells, recombinant cells, or recombinant microorganisms.
Further, the recombinant vector of the present disclosure may include a selection marker, which is for selecting a transformant (host cell) transformed with the vector. In a medium treated with the selection marker, only cells expressing the selection marker may survive, and thus transformed cells may be selected. The selection marker may be represented by kanamycin, streptomycin, chloramphenicol, etc., but is not limited thereto.
The genes inserted into the recombinant vector for transformation of the present disclosure may be introduced into host cells such as microorganisms of the genus Corynebacterium due to homologous recombination crossover.
According to a specific embodiment of the present disclosure, the host cell may be a strain of the genus Corynebacterium, for example, Corynebacterium glutamicum strain.
Further, another aspect of the present disclosure provides a method of producing L-lysine, the method including the steps of a) culturing the Corynebacterium glutamicum variant in a medium; and b) recovering L-lysine from the variant or the medium in which the variant is cultured.
The culturing may be performed according to a suitable medium and culture conditions known in the art, and any person skilled in the art may easily adjust and use the medium and culture conditions. Specifically, the medium may be a liquid medium, but is not limited thereto. The culturing method may include, for example, batch culture, continuous culture, fed-batch culture, or combinations thereof, but is not limited thereto.
According to a specific embodiment of the present disclosure, the medium should meet the requirements of a specific strain in a proper manner, and may be appropriately modified by a person skilled in the art. For the culture medium for the strain of the genus Corynebacterium, reference may be made to a known document (Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington D.C., USA, 1981), but is not limited thereto.
According to a specific embodiment of the present disclosure, the medium may include various carbon sources, nitrogen sources, and trace element components. Carbon sources that may be used include saccharides and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, cellulose, etc., oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, etc., fatty acids such as palmitic acid, stearic acid, and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These substances may be used individually or in a mixture, but are not limited thereto. Nitrogen sources that may be used include peptone, yeast extract, meat extract, malt extract, corn steep liquor, soybean meal, urea, or inorganic compounds, e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. The nitrogen sources may also be used individually or in a mixture, but are not limited thereto. Phosphorus sources that may be used may include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts, but are not limited thereto. Further, the culture medium may include metal salts such as magnesium sulfate or iron sulfate, which are required for growth, but is not limited thereto. In addition, the culture medium may include essential growth substances such as amino acids and vitamins. Moreover, suitable precursors may be used in the culture medium. The medium or individual components may be added to the culture medium batchwise or in a continuous manner by a suitable method during culturing, but are not limited thereto.
According to a specific embodiment of the present disclosure, pH of the culture medium may be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the microorganism culture medium in an appropriate manner during the culturing. In addition, during the culturing, foaming may be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. Additionally, to keep the culture medium in an aerobic condition, oxygen or an oxygen-containing gas (e.g., air) may be injected into the culture medium. The temperature of the culture medium may be generally 20° C. to 45° C., for example, 25° C. to 40° C. The culturing may be continued until a desired amount of the useful substance is produced. For example, the culturing time may be 10 hours to 160 hours.
According to a specific embodiment of the present disclosure, in the step of recovering L-lysine from the cultured variant and the medium in which the variant is cultured, the produced L-lysine may be collected or recovered from the culture medium using a suitable method known in the art depending on the culture method. 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), etc. may be used, but the method is not limited thereto.
According to a specific embodiment of the present disclosure, in the step of recovering lysine, the culture medium is centrifuged at a low speed to remove biomass, and the obtained superatant may be separated through ion exchange chromatography.
According to a specific embodiment of the present disclosure, the step of recovering L-lysine may include a process of purifying L-lysine.
A Corynebacterium glutamicum variant according to the present disclosure may improve a production yield of L-lysine by increasing or enhancing the expression of a gene encoding aspartate aminotransferase, as compared to a parent strain.
Hereinafter, the present disclosure will be described in more detail. However, this description is merely provided to aid understanding of the present disclosure, and the scope of the present disclosure is not limited by this exemplary description.
To prepare a Corynebacterium glutamicum variant with the enhanced aspartate aminotransferase activity, Corynebacterium glutamicum DS1 strain was used to induce random mutation.
Corynebacterium glutamicum DS1 strain was inoculated in a flask containing 50 ml of a CM liquid medium (5 g of glucose, 2.5 g of NaCl, 5.0 g of yeast extract, 1.0 g of urea, 10.0 g of polypeptone and 5.0 g of beef extract, pH 6.8), and N-methyl-N′-nitro-N-nitrosoguanidine (NTG), which is a mutagen, was added at a final concentration of 300 μg/ml, followed by culturing at 30° C. with shaking at 200 rpm for 20 hours. Then, to induce additional mutation, UV exposure was performed for 20 minutes. After completion of the culturing, the culture was centrifuged at 12,000 rpm for 10 minutes to remove the supernatant, and the resultant washed once with saline, and washed three times or more with phosphate buffer. This was suspended in 5 ml of phosphate buffer, spread on a CM solid medium (15 g/l agar and 8% lysine was further added to CM liquid medium), and cultured at 30° C. for 30 hours to isolate 100 colonies.
1-2. Selection of Variants with Improved L-Lysine Producing Ability
Each 5% of 100 isolated colonies was inoculated into a flask containing 10 ml of a production liquid medium shown in Table 1 below, and cultured with shaking at 200 rpm at 30° C. for 30 hours. Absorbance of each culture was measured at OD 610 nm, and the L-lysine production was compared to select 10 colonies producing 75.0 g/l or more of L-lysine, as compared to Corynebacterium glutamicum DS1 strain, in which mutation was not induced, and the nucleotide sequences thereof were analyzed to identify mutation sites in the promoter of aspB gene. As a result of examining the nucleotide sequences of the Corynebacterium glutamicum DS1 variants, in which these mutations were induced, three types of mutations were identified: C> G at position −22, T> A at position −45, and T> A at position −88.
Thereafter, an experiment was performed to examine an increase in the L-lysine productivity due to three types of promoter mutations of the aspB gene.
To prepare a Corynebacterium glutamicum variant with the enhanced aspartate aminotransferase activity, Corynebacterium glutamicum DS1 strain and E. coli DH5a (HIT Competent cells™, Cat No. RH618) were used.
The Corynebacterium glutamicum DS1 strain was cultured at a temperature of 30° C. in a CM liquid or solid medium (adding 15 g/L of agar, as needed) (pH 6.8) having a composition of 5 g of glucose, 2.5 g of NaCl, 5.0 g of yeast extract, 1.0 g of urea, 10.0 g of polypeptone, and 5.0 g of a beef extract in 1 L of distilled water.
The E. coli DH5a was cultured at a temperature of 37° C. in an LB medium having a composition of 10.0 g of tryptone, 10.0 g of NaCl, and 5.0 g of a yeast extract in 1 L of distilled water.
Antibiotics, kanamycin and streptomycin, products of Sigma, were used, and DNA sequencing analysis was conducted at Macrogen Co., Ltd.
To increase lysine productivity by enhancing the supply of a lysine precursor aspartate in the strain, it was intended to enhance the activity of aspartate aminotransferase. The method used in this Example induced a specific mutation in a promoter of aspB gene in order to increase expression of aspB gene encoding aspartate aminotransferase. The nucleotide sequence at position −22 of the promoter of the aspB gene was replaced with G, and a fragment of 1256 bp around the aspB gene on the genome of each variant obtained in Example 1 was amplified by PCR, and cloned into a recombinant vector pCGI (see [Kim et al., Journal of Microbiological Methods 84 (2011) 128-130]). The plasmid was named pCGI(DS7-2) (see
PCR was performed using the above primers under the following conditions. 25 to 30 cycles were performed using a thermocycler (TP600, TAKARA BIO Inc., Japan) in the presence of 1 unit of pfu-X DNA polymerase mix (Solgent) using 1 pM of oligonucleotide and 10 ng of chromosomal DNA of Corynebacterium glutamicum DS1 variant (mutation occurred at position −22 of the promoter) selected in Example 1 as a template in a reaction solution to which 100 UM of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, dTTP) was added. PCR was performed under conditions of (i) denaturation step: at 94° C. for 30 seconds, (ii) annealing step: at 58° C. for 30 seconds, and (iii) extension step: at 72° C. for 1 minute to 2 minutes (polymerization time of 2 minutes per 1 kb).
Each gene fragment thus prepared was cloned into the pCGI vector using self-assembly cloning. The vector was transformed into E. coli DH5a, plated on an LB-agar plate containing 50 μg/mL kanamycin, and cultured at 37° C. for 24 hours. The finally formed colonies were isolated, and it was confirmed whether the insert was correctly present in the vector, which was isolated and used in the recombination of Corynebacterium glutamicum strain.
DS7-2 strain which is a strain variant was prepared using the pCGI(DS7-2) vector. The vector was prepared at a final concentration of 1 μg/μL or more, and primary recombination was induced in the Corynebacterium glutamicum DS1 strain using electroporation (see a document [Tauch et al., FEMS Microbiology letters 123 (1994) 343-347]). At this time, the electroporated strain was then spread on a CM solid medium containing 20 μg/μL kanamycin to isolate colonies, and then it was confirmed through PCR and base sequencing analysis whether the vector was properly inserted into the induced position on the genome. To induce secondary recombination, the isolated strain was inoculated into a CM liquid medium, cultured overnight or longer, and then spread on an agar medium containing 40 mg/L of the same concentration of streptomycin to isolate colonies. After examining kanamycin resistance in the finally isolated colonies, it was confirmed through base sequencing analysis whether mutations were introduced into the promoter of the aspB gene in strains without antibiotic resistance (see a document [Schafer et al., Gene 145 (1994) 69-73]). Finally, Corynebacterium glutamicum variant (DS7-2), into which the mutated aspB promoter was introduced, was obtained.
A Corynebacterium glutamicum variant was prepared in the same manner as Example 2, except that the nucleotide sequence at the position −45 of the promoter of the aspB gene was replaced from T to A, and Corynebacterium glutamicum DS1 variant (mutation occurred at position −45 of the promoter) selected in Example 1 as a DNA template was used.
Here, to construct the plasmid, primers shown in Table 2 below were used to amplify each gene fragment, and DS7-1 strain which is a strain variant was prepared using the prepared plasmid pCGI(DS7-1) vector (see
A Corynebacterium glutamicum variant was prepared in the same manner as Example 2, except that the nucleotide sequence at the position −88 of the promoter of the aspB gene was replaced from T to A, and Corynebacterium glutamicum DS1 variant (mutation occurred at position −88 of the promoter) selected in Example 1 as a DNA template was used.
Here, to construct the plasmid, primers shown in Table 2 were used to amplify each gene fragment, and DS7 strain which is a strain variant was prepared using the prepared plasmid pCGI(DS7) vector (see
The L-lysine productivity was compared between the parent strain Corynebacterium glutamicum DS1 strain, DS7-2, DS7-1 and DS7 strains which are the lysine producing variants prepared in Examples 2 to 4.
Each strain was inoculated into a 100 ml flask containing 10 ml of a lysine medium having the composition as in Table 1, and cultured with shaking at 180 rpm at 30° C. for 28 hours. After completion of the culturing, L-lysine analysis was performed by measuring the L-lysine production using HPLC (Shimazu, Japan). The results are shown in Table 3.
As shown in Table 3, it was confirmed that Corynebacterium glutamicum variants, DS7-2, DS7-1 and DS7 strains, exhibited about 3.7%, 7.7%, and 16.9% increases in the L-lysine productivity, respectively, as compared to the parent strain Corynebacterium glutamicum DS1 strain, due to substitution of the specific positions (−22, −45, or −88 regions) in the promoter sequence of the aspB gene with the optimal nucleotide sequence for strengthening the lysine biosynthetic pathway. In particular, it was confirmed that DS7 strain exhibited the highest productivity, as compared to other variants. These results indicate that the enhanced expression of the aspB gene may promote the supply of lysine precursor, thereby improving the L-lysine producing ability of the strain.
Hereinabove, the present disclosure has been described with reference to preferred exemplary embodiments thereof. It will be understood by those skilled in the art to which the present disclosure pertains that the present disclosure may be implemented in modified forms without departing from the essential characteristics of the present disclosure. Accordingly, exemplary embodiments disclosed herein should be considered in an illustrative aspect rather than a restrictive aspect. The scope of the present disclosure is shown not in the aforesaid explanation but in the appended claims, and all differences within a scope equivalent thereto should be interpreted as being included in the present disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2021-0055536 | Apr 2021 | KR | national |
| 10-2021-0066151 | May 2021 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2021/006499 | 5/25/2021 | WO |
