KR101694811B1 - Mutant Strain with improved Amino acid production by inactivating malic enzyme or lactate dehydrogenase - Google Patents

Abstract

The present invention relates to a mutant strain of arginine or glutamine production including a malic enzyme or a lactate dehydrogenase, and a method for producing the same. According to the present invention, by inactivating a malic acid enzyme or a lactate dehydrogenase, it is possible to provide a mutant having an improved arginine or glutamine production ability and yield per unit without lowering the growth of an arginine or glutamine producing ability strain including a wild type or an activated malic acid enzyme or a lactate dehydrogenase A strain can be produced to produce arginine or glutamine. In addition, since the mutant strains of the present invention can be used to improve the yield and yield per unit of arginine or glutamine, there is an advantage in cost reduction when arginine or glutamine is used as a raw material.

Description

Mutant Strain with improved Amino acid production by inactivating malic enzyme or lactate dehydrogenase}

The present invention relates to a mutant strain having improved amino acid production ability by inactivating malic enzyme or lactate dehydrogenase.

L-Arginine is a kind of natural amino acid that is widely used in medicines, foods, and other animal feeds. For medicine, it is used as a liver function stimulator, brain function stimulator, male infertility treatment, and comprehensive amino acid preparation. It is a substance that has recently been in the spotlight for adding health drinks and as a substitute for salt in hypertensive patients.

L-glutamine is a type of L-glutamic acid-based amino acid, and as it has been confirmed to be effective in cell regeneration in recent years, demand is increasing as a cosmetic ingredient, and research on anti-aging is continuing. In addition, L-glutamine has been steadily used in medicine, such as nutritional supplements, gastrointestinal ulcer treatment, alcoholism treatment, and brain function improvement agents. On the other hand, L-glutamine was originally classified as not an essential amino acid, but in recent years it has been identified as a Conditional Essential Amino acid that has the functionality of an essential amino acid, and its importance is being further emphasized.

Methods for developing amino acid-producing strains have been various from the prior art. The most common amino acid production strain development methods include selection of resistant strains for intermediate metabolites or final products using a mutation method, and activation, deletion or enhancement of genes that regulate the production of target products through chromosomal mutation. That is, a method of selecting a mutant strain having resistance to an amino acid-like structure can be applied by using the mutation method. In addition, there are methods of reducing or blocking the activity of biosynthetic pathways corresponding to branch points other than the target product, or enhancing the biosynthetic pathway to the target product. . However, in this case, unwanted mutations may occur, resulting in a problem in which production of the target product is reduced due to growth reduction and feedback inhibition.

The malE gene deleted in the present invention is a gene encoding malic enzyme, an enzyme involved in converting malate to pyruvate, and ldhA gene converts pyruvate to lactate. It is a gene that codes for lactate dehydrogenase, an enzyme involved in the process. The present inventors tried to increase the carbon flow in the direction of the TCA ring from pyruvate by inactivating the malic enzyme or lactate dehydrogenase, thereby finally increasing the productivity of arginine or glutamine (see FIG. 1).

Throughout this specification, a number of papers and patent documents are referenced and citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated herein by reference as a whole, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

The present inventors have made intensive research efforts to develop a method for increasing the productivity of arginine or glutamine. As a result, when the malic enzyme or lactate dehydrogenase is inactivated using genetic recombination technology, the present invention was completed by confirming that the productivity and sugar yield of arginine or glutamine can be increased.

Accordingly, an object of the present invention is to provide an arginine or glutamine-producing mutant strain comprising inactivated malic enzyme or inactivated lactic acid dehydrogenase.

Another object of the present invention is to provide a method for producing an arginine or glutamine-producing mutant strain comprising the step of inactivating malic enzyme or lactate dehydrogenase.

Another object of the present invention is to provide a method for producing arginine or glutamine comprising the step of culturing the mutant strain of the present invention.

Other objects and advantages of the present invention will become more apparent by the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention provides an arginine or glutamine-producing mutant strain comprising inactivated malic enzyme or inactivated lactate dehydrogenase.

The present inventors have made intensive research efforts to develop a method for increasing the productivity of arginine or glutamine. As a result, it was confirmed that when the malic enzyme or lactate dehydrogenase is inactivated using gene recombination technology, the productivity and the yield of arginine or glutamine can be increased.

In the present invention, the term “inactivation” used while expressing an arginine or glutamine-producing mutant strain refers to suppressing the expression of a protein by substitution, insertion, deletion, or a combination of nucleotides encoding genes.

According to one embodiment of the present invention, the inactivation of the malic enzyme of the present invention is inactivation by substitution, insertion, deletion, or a combination of the nucleotide sequence encoding the malE gene. According to another embodiment of the present invention, the inactivation of the malic enzyme of the present invention is inactivation by complete or partial deletion of the nucleotide sequence encoding the malE gene. According to a specific embodiment of the present invention, the inactivation of the malic enzyme of the present invention is inactivation by partial deletion of the nucleotide sequence encoding the malE gene.

According to an embodiment of the present invention, the inactivation of the lactate dehydrogenase of the present invention is ldhA It is inactivation by substitution, insertion, deletion, or a combination of the nucleotide sequence encoding the gene. According to another embodiment of the present invention, the inactivation of the lactate dehydrogenase of the present invention is the inactivation of lactate dehydrogenase by complete or partial deletion of the nucleotide sequence encoding the ldhA gene. According to a specific embodiment of the present invention, the inactivation of the lactate dehydrogenase of the present invention is ldhA It is the inactivation of lactate dehydrogenase by partial deletion of the nucleotide sequence encoding the gene.

The mutant strain of the present invention is a strain in which malic enzyme or lactate dehydrogenase is inactivated by using an arginine or glutamine-producing strain containing wild type or activated malic enzyme as a parent strain, and expressing malic enzyme or lactate dehydrogenase. Or, any strain containing the enzymatic activity of malic enzyme or lactate dehydrogenase can be used without limitation as a parent strain.

According to one embodiment of the present invention, the arginine-producing mutant strain of the present invention is the genus Corynebacterium or the genus Brevibacterium. According to another embodiment of the present invention, the arginine-producing mutant strain of the present invention is Corynebacterium glutamicum , Corynebacterium acetoglutamicum acetoglutamicum ), Corynebacterium acetoacedophilum ( Corynebacterium acetoacidophilum ), Corynebacterium thermoaminogenes ( Corynebacterium thermoaminogenaes ), Brevibacterium flavum (Brevibacterium flavum ), Brevibacterium lactofermentum ( Brevibacterium lactofermentum ), Brevibacterium divaricatum . According to a specific embodiment of the present invention, the arginine-producing mutant strain of the present invention is Corynebacterium glutamicum. According to another embodiment of the present invention, the Corynebacterium glutamicum is Corynebacterium glutamicum ( Corynebacterium glutamicum ) KCTC 10423BP.

According to a specific embodiment of the present invention, the arginine-producing mutant strain of the present invention is Corynebacterium acetoglutamicum ATCC15806, Corynebacterium acetoacedophilum ATCC13870, Corynebacterium thermoaminogenes FERM BP-1539, Brevibacterium flavum ATCC14067, Brevibacterium lactofermentum ATCC13869, Brevibacterium divaricatum ATCC14020.

According to an embodiment of the present invention, the glutamine-producing mutant strain of the present invention is Corynebacterium genus, Brevibacterium genus, Micrococcus genus, Microbacterium genus , Bacillus genus, Streptomyces genus, Phenylcilium genus, Pseudomonas genus, Arthrobacter genus, Serratia genus, Candida genus, Aerobacter aerogenes, Kleb It is a strain selected from the group consisting of the genus Klebsiella, the genus Erwinia, and the genus Pantoea. According to another embodiment of the present invention, the glutamine-producing mutant strain of the present invention is a genus Corynebacterium, a genus Brevibacterium, or a genus Microbacterium. According to a specific embodiment of the present invention, the glutamine-producing mutant strain of the present invention is Corynebacterium acetoaxidofilum (Corynebacterium acetoacidophilum ), Corynebacterium acetoglutamicum (Corynebacterium acetoglutamicum ), Corynebacterium alkanolyticum , Corynebacterium callunae ), Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium lilium , Corynebacterium melasecola (Corynebacterium melassecola ), Corynebacterium thermoaminogenes ( Corynebacterium thermoaminogenes), Corynebacterium epi when Enschede (Corynebacterium efficiens), Corynebacterium Herr cool-less (Corynebacterium herculis ), Brevibacterium divaricatum (Brevibacterium divaricatum ), Brevibacterium flavum (Brevibacterium flavum ), Brevibacterium immariophilum ( Brevibacterium immariophilum), Brevibacterium lactofermentum (Brevibacterium lactofermentum), erecting a Brevibacterium (Brevibacterium roseum ), Brevibacterium saccharolithcum ( Brevibacterium saccharolyticum), Brevibacterium bring you tea Tallis (Brevibacterium thiogenitalis), Brevibacterium ammonia Ness's (Brevibacterium ammoniagenes ), Brevibacterium albumin ( Brevibacterium album ), Brevibacterium serinum ( Brevibacterium cerinum ), Microbacterium ammoniaphilum . According to any embodiment of the invention, glutamine producing ability mutant strain of the present invention is Corynebacterium acetonitrile solution attempts pilreom, Corynebacterium acetonitrile glutamicum, Corynebacterium alkanoyl utility glutamicum, Corynebacterium Kalou I, Corynebacterium glutamicum, Corynebacterium Lilium, Corynebacterium melanoma theta coke, Corynebacterium thermo amino to Ness, Corynebacterium epi when Enschede, Corynebacterium Herr cool is less . According to another embodiment of the present invention, the glutamine-producing mutant strain of the present invention is Corynebacterium glutamicum ( Corynebacterium glutamicum ). According to a specific embodiment of the present invention, the glutamine-producing mutant strain of the present invention is Corynebacterium glutamicum accession number KFCC 10694.

According to an embodiment of the present invention, the arginine-producing mutant strain containing the inactivated malic enzyme of the present invention has an arginine-producing ability of 3-30%, 5 as compared to the wild-type or activated malic enzyme-containing arginine-producing strain. -30%, 7-30%, 9-30%, 9-25%, 9-23% or 9-21% increased strain. According to another embodiment of the present invention, the increase in the arginine production ability is 3-20%, 5-20%, 7-20%, 9-20%, 9-17%, 9-15%, 9- This is an increase of 13% or 9-11%. According to another embodiment of the present invention, the increase in the arginine production ability is 10-30%, 15-30%, 17-30%, 19-30%, 19-25%, 19-23% when cultured in a 5 L culture tank. Or a 19-21% increase.

According to an embodiment of the present invention, the arginine-producing mutant strain containing the inactivated malic enzyme of the present invention has a per-unit yield of 3-30% of arginine compared to the arginine-producing strain containing the wild-type or activated malic enzyme, 5-30%, 7-30%, 9-30%, 9-25%, 9-23% or 9-21% increased strain. In the present specification, the term "unit yield" refers to the ratio of the production amount (weight/weight) of arginine or glutamine to glucose used as a carbon source and an energy source. According to another embodiment of the present invention, the increase in the glucose yield is 3-20%, 5-20%, 7-20%, 9-20%, 9-17%, 9-15% or 9-13 during flask culture. % Increase. According to another embodiment of the present invention, the increase in the glucose yield is 10-30%, 15-30%, 17-30%, 19-30%, 19-25%, 19-23% or That's a 19-21% increase.

According to one embodiment of the present invention, the glutamine-producing mutant strain containing the inactivated malic enzyme of the present invention has a glutamine-producing ability of 3-30% compared to the glutamine-producing strain containing the wild-type or activated malic enzyme, 3 -20%, 5-20%, 5-15% or 5-10% increased strain.

According to an embodiment of the present invention, the glutamine-producing mutant strain containing the inactivated malic enzyme of the present invention has a glucose yield of 3-30% compared to the glutamine-producing ability strain containing the wild-type or activated malic enzyme, 3-20%, 5-20%, 5-15% or 5-10% increased strain.

According to an embodiment of the present invention, the arginine-producing mutant strain containing the inactivated lactate dehydrogenase of the present invention has an arginine-producing ability of 3-20% compared to the wild type or the arginine-producing ability strain containing the activated lactate dehydrogenase. , 5-20%, 7-20%, 9-20%, 9-17%, 9-15% or 9-13% increased strain.

According to an embodiment of the present invention, the arginine-producing mutant strain comprising the inactivated lactate dehydrogenase of the present invention has a per-unit yield of 3-20 compared to the arginine-producing strain comprising the wild-type or activated lactate dehydrogenase. %, 5-20%, 8-20%, 8-17%, 8-15% or 8-12% increased strain.

According to one embodiment of the present invention, the glutamine-producing mutant strain containing the inactivated lactate dehydrogenase of the present invention has a glutamine-producing ability of 3-30% compared to the glutamine-producing ability strain containing the wild-type or activated lactate dehydrogenase. , 5-30%, 7-30%, 7-25%, 7-20%, 7-17% or 7-14% increased strain. According to another embodiment of the present invention, the increase in glutamine production capacity is 5-20%, 7-20%, 10-20%, 10-17% or 10-15% increase in flask culture. According to another embodiment of the present invention, the increase in glutamine production capacity is 3-20%, 5-20%, 5-15%, 7-15%, or 7-10% increase when cultured in a 5 L culture tank.

According to one embodiment of the present invention, the glutamine-producing mutant strain containing the inactivated lactate dehydrogenase of the present invention has a glucose yield of 5-30% compared to the glutamine-producing strain containing the wild-type or activated malic enzyme. , 10-30%, 12-30%, 12-25%, 12-20% or 12-17% increased strain.

According to another aspect of the present invention, the present invention provides a method for producing an arginine or glutamine-producing mutant strain comprising the step of inactivating malic enzyme or lactate dehydrogenase.

In the present invention, it is possible to inactivate malic enzyme or lactic acid dehydrogenase by a known gene recombination method in an arginine or glutamine-producing strain containing wild-type or activated malic enzyme or lactate dehydrogenase.

The recombinant plasmid pK19mobsacB::Δ malE or pK19mobsacB::Δ ldhA is used to delete the gene malE encoding the malic enzyme involved in arginine or glutamine biosynthesis or the gene ldhA encoding the lactate dehydrogenase from the chromosome.

In the present invention, the step of transforming an arginine or glutamine-producing strain containing wild-type or activated malic enzyme or lactate dehydrogenase may be performed through a known method. For example, it may be carried out by an electroporation method (van der Rest et al., Appl. Microbiol. Biotechnol., 52, 541-545, 1999).

In addition, the present invention includes the step of isolating and obtaining a transformed mutant strain from an arginine or glutamine-producing strain containing untransformed wild-type or activated malic enzyme or lactate dehydrogenase.

In the present invention, a selectively labeled vector may be used in performing the step of separating and obtaining the transformed mutant strain from the untransformed arginine or glutamine-producing strain strain.

In addition, the vector used in the present invention includes, but is not limited to, a plasmid or phage.

Selective markers used in the present invention include antibiotic resistance genes such as kanamycin and chloramphenicol commonly used in the art, and levansucrase, which is a product of the SacB gene derived from Bacillus bacillus.

The production method of the present invention is a method of producing the same strain as the mutant strain of arginine or glutamine-producing ability containing the above-described inactivated malic enzyme or lactate dehydrogenase, and proteins to be inactivated (malic enzyme or lactate dehydrogenase) And related genes ( malE Or, since ldhA ) is common, descriptions of the common content between the two are omitted in order to avoid undue complexity of the present specification.

According to another aspect of the present invention, the present invention provides a method for producing arginine or glutamine comprising culturing the mutant strain of the present invention.

The present invention can be cultured through known culture methods in pre-culture or seed culture of arginine or glutamine-producing strains and mutant strains.

Natural medium or synthetic medium can be used in the present invention, and the carbon source of the medium may be, for example, glucose, sucrose, dextrin, glycerol, starch, etc., and as the nitrogen source, peptone, meat extract, yeast extract, dried Yeast, soybean cake, urea, thiourea, ammonium salts, nitrates and other organic or inorganic nitrogen-containing compounds may be used, but are not limited to these ingredients.

Inorganic salts contained in the medium may include phosphates such as magnesium, manganese, potassium, calcium, iron, etc., nitrates, carbonates, chlorides, etc., but are not limited thereto.

In addition to the components of the carbon source, nitrogen source, and inorganic salt, amino acids, vitamins, nucleic acids, and compounds related thereto may be added to the medium.

The present invention is a method of producing arginine or glutamine by using the same strain and strain manufacturing method as the arginine or glutamine-producing mutant strain containing the inactivated malic enzyme or lactate dehydrogenase. In order to avoid excessive complexity, the description is omitted.

According to another aspect of the present invention, the present invention provides arginine or glutamine produced using the method of the present invention.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides an arginine or glutamine-producing mutant strain containing inactivated malic enzyme or lactate dehydrogenase, and a method for producing the same.

(b) The present invention provides a method for producing arginine or glutamine comprising the step of culturing the mutant strain of the present invention.

(c) In the present invention, by inactivating malic enzyme or lactate dehydrogenase, arginine or glutamine-producing ability and sugar yield are not lowered than that of arginine or glutamine-producing strains containing wild-type or activated malic enzyme or lactate dehydrogenase. There is an effect capable of producing arginine or glutamine by preparing an improved mutant strain.

(d) In the present invention, it is possible to improve the production amount and yield of arginine or glutamine by using the mutant strain of the present invention, and there is an advantage of cost reduction when arginine or glutamine is used as a raw material.

1 is malE Gene and ldhA It represents the pathway through which the gene is involved.
Figures 2a to 2d are malE Gene or ldhA It shows the PCR result confirming the defective part of the gene. Figure 2a is AR ( malE : 2913 bp) and AR-Δ malE (Δ malE : 1854 bp) , Figure 2b is 365 ( malE : 2739 bp) and 365-Δ malE (Δ malE : 1693 bp) , Figure 2c is AR ( ldhA : 2890 bp) and AR-Δ ldhA (Δ ldhA : 2101 bp) , Figure 2d shows the results confirmed in 365 (ldhA : 2812 bp) and 365-Δ ldhA (Δ ldhA : 2066 bp).
Figures 3a and 3b show the amount of arginine production and yield in a flask (Fig. 3a) and a 5L fermenter (Fig. 3b) of the parent strain and the strain in which the malE gene has been deleted.
Figures 4a and 4b show the amount of glutamine production and the unit yield in flasks (Figure 4a) and 5 L fermenters (Figure 4b) of the parent strain and the strain in which the malE gene has been deleted.
5A and 5B show the amount of arginine production and the yield per unit in the flask (FIG. 5A) and 5L fermenter (FIG. 5B) of the parent strain and the strain in which the ldhA gene was deleted.
6 is a parent strain and ldhA It shows the amount of lactic acid produced in the flask of the strain from which the gene was deleted.
7A and 7B show the amount of glutamine production and the yield per unit in flasks (FIG. 7A) and 5L fermenters (FIG. 7B) of the parent strain and the strain in which the ldhA gene was deleted.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Throughout this specification, “%” used to indicate the concentration of a specific substance is (weight/weight)% for solids/solids, (weight/volume)% for solids/liquids, and Liquid/liquid is (vol/vol) %.

Example 1: malE Genes and ldhA Manufacture of mutant strains with deletion of genes

The malE gene or the ldhA gene was deleted by homologous recombination. The nucleotide sequence of the malE gene or ldhA gene to be deleted is SEQ ID NO: 1 ( malE nucleotide sequence of Corynebacterium glutamicum AR), SEQ ID NO: 2 ( malE nucleotide sequence of Corynebacterium glutamicum 365), SEQ ID NO: 3 (LdhA nucleotide sequence of Corynebacterium glutamicum AR) and SEQ ID NO: 4 ( LdhA nucleotide sequence of Corynebacterium glutamicum 365).

Using the Wizard Genomic DNA Purification Kit (Promega, USA), the glutamine-producing strain Corynebacterium glutamicum 365 (Accession No. KFCC 10694) and the arginine-producing strain Corynebacterium Glue Tamicum AR (Corynebacterium glutamicum AX40-34 (Accession No. KCTC 10423BP)) was subjected to mutation treatment (e.g., UV irradiation or MNNG (N-methyl-N'-nitrosoguanidine) treatment) and arginine hydroxamate (L -Isolation of chromosomal DNA (genomic DNA) of Corynebacterium glutamicum AR (arginine producing strain) and Corynebacterium glutamicum 365 (glutamine producing strain) resistant to Arginie hydroxamate hydrochloride, Sigma, USA I did. The method for separating a strain resistant to arginine hydroxamate by mutating the arginine-producing strain is described in detail in Korean Patent No. 10-0052645 and Korean Patent No. 10-0055145 of the present inventors, which Incorporated herein by reference.

The chromosomal DNA was used as a template by polymerase chain reaction (PCR) to obtain the following deleted PCR products: 803 bp malE DNA fragment 1 and 803 bp fragment 2 (template: Coryne Bacterium glutamicum AR); 719 bp malE DNA fragment 1 and 671 bp fragment 2 (template: Corynebacterium glutamicum 365); 925 bp ldhA DNA fragment 1 and 906 bp fragment 2 (template: Corynebacterium glutamicum AR); And 917 bp ldhA DNA fragment 1 and 898 bp fragment 2 (template: Corynebacterium glutamicum 365).

PCR reaction (total reactions) using the primer pairs (1-F/1-R set, 2-F/2-R set) in Table 1 below to perform the primary PCR for constructing malE or ldhA deletion strains. The volume was 50 μL, 94° C. for 5 minutes and 1 cycle, 94° C. for 30 seconds, 55° C. for 30 seconds, 72° C. for 1 minute and 30 seconds, for a total of 30 cycles, followed by 72° C. for 5 minutes). The annealing temperature was adjusted between 55°C and 62°C based on the Tm value of the primer, and the elongation time was adjusted based on 2 minutes per 1 kb according to the size of the PCR fragment.

After obtaining the first PCR product, as the second PCR, overlap PCR was performed using fragment 1 and fragment 2 as a template, and the final PCR product, A-Δ malE DNA fragment, 1606 bp (template: Corynebacterium glutamicum AR), G-Δ malE DNA fragment 1390 bp (template: Corynebacterium glutamicum 365) was obtained, and A-Δ ldhA DNA fragment 1831 bp (template: Corynebacterium glutamicum AR), G- Δ ldhA DNA fragment 1702 bp (template: Corynebacterium glutamicum 365) was amplified.

Thereafter, each PCR product was digested by reacting at 37° C. for 2 hours using each restriction enzyme.

For the final vector preparation, the pK19mobsacB vector was also treated with the same restriction enzyme and then ligated. The ligation reaction was performed using ligation kit version 2.1 (Ligation kit Ver 2.1, Takara, Japan).

The completed vector was introduced into arginine or glutamine-producing strains by electroporation. The final candidates AR-Δ malE, 365-Δ malE, AR-Δ ldhA and 365-Δ ldhA are the primer pairs in Table 1 (A -malE -cf and A -malE -cr or A- ldhA -cf and A- ldhA -cr , for glutamine-producing strains, use G- malE -cf and G- malE -cr or G- ldhA -cf and G -ldhA -cr) to determine the malE gene or ldhA gene. It was confirmed whether it was deleted (FIGS. 2A to 2D ).

Arginine was diluted 100 times with distilled water, filtered through a 0.45 μm filtration membrane, and then used high-performance liquid chromatography (HPLC) equipped with a column (Universal Cation HR 3u, 100 mm, Part No. 23100) and an ultraviolet detector (195 nm). Analyzed.

Glutamine was analyzed using high-performance liquid chromatography (HPLC) equipped with a column (Apollo C18 5mm Part No. 36515 length 150mm ID 4.6) UV detector (210nm) after diluting the culture medium 100 times with distilled water and filtering through a 0.45 μm filtration membrane. I did.

* Capital letters indicate the restriction enzyme part

Example 2: Arginine Production strain AR and malE gene Fruiting bacteria Arginine production evaluation (flask)

MalE obtained in Example 1 above In order to confirm the amount of arginine production of the gene-deleting strain, the amount of arginine production of the parent strain, the arginine-producing strain AR, and the malE gene-deleted strain, AR- malE , were compared. Arginine production was compared by flask titer experiments in the medium consisting of the composition of Table 2 for the two strains.

A flask titer experiment was performed under the medium conditions, and the physical culture conditions were incubated for 44 hours at a temperature of 30° C. and a stirring speed of 140 rpm. Table 3 below relates to a comparison of the amount of arginine produced by the parent strain and the malE deletion strain (flask titer experiment).

As shown in the results of Table 3, the AR-Δ malE strain was found to have increased production of arginine compared to the parent strain AR, resulting in a higher yield per unit. Arginine production from AR and AR-Δ malE culture results are compared with relative values, and when the production amount of the parent strain is 100%, the production amount of AR-Δ malE is 110%, and the yield per unit is 100% per unit yield of the parent strain. It was confirmed that both the production amount of arginine and the yield per unit were increased to 111% by the present invention (FIG. 3A).

Example 3: Arginine Production strain AR Wow malE gene Fruiting bacteria Arginine production evaluation (fermentation tank)

Arginine-producing strains AR and malE in a 5 L fermenter consisting of the medium composition in Table 4 The amount of arginine production of gene deletion strains was compared.

As for the injection and additional medium, A, B, C, and D were sterilized separately, and 275 ml of the seed culture was inoculated as a culture condition, and the amount of the injection liquid was 1.65 L, a temperature of 30° C., an agitation speed of 500 rpm, and It was incubated under the physical conditions of an aeration amount of 1.0 vvm. When the amount of sugar reached 1.5%, the additional medium was added aseptically, and the additional medium was added a total of 7 times. AR-malE obtained in the present invention was cultured under the same conditions as the parent strain. Table 6 below relates to a comparison of the amount of arginine production (5 LJ/F) of the parent strain and malE deletion strain.

As shown in the results of Table 6, the AR-Δ malE strain was found to increase the production of arginine without reducing the growth compared to the parent strain, resulting in a higher yield per unit. All of the arginine and substitute yields in Table 6 are relative values. As a result of culturing AR-Δ malE for the same time as the parent strain (81-84 hr), the result showed an OD value of 57.5, showing a similar growth degree to that of the parent strain. Further, the AR-Δ malE arginine yield is 120% when the production of the parental strain of 100%, per the yield also AR-Δ malE by the invention is 120% when the per yield of parent strain to 100% of the It was confirmed that both the production amount of arginine and the yield per unit were increased (Fig. 3b).

Example 4: glutamine Production strain 365 and malE gene Fruiting bacteria Evaluation of glutamine production (flask)

In order to confirm the glutamine production of the malE gene deletion strain obtained in Example 1, the parent strain, glutamine-producing strain 365 and malE The amount of glutamine production of 365-malE , a strain in which the gene was deleted, was compared. Glutamine production was compared by flask titer experiments in the medium consisting of the composition of Table 7 for the two strains.

A flask titer experiment was performed under the above medium conditions, and the physical culture conditions were incubated for 40 hours at a temperature of 30° C. and a stirring speed of 140 rpm. Table 8 below relates to a comparison of the amount of glutamine produced by the parent strain and the malE deletion strain (flask titer experiment).

As shown in the results of Table 8, the 365-Δ malE strain was found to have increased glutamine production compared to the parent strain, resulting in an increased yield. When comparing the glutamine production results of 365 and 365-Δ malE strain cultivation as a relative value, when the production of the parent strain is 100%, the yield of 365-Δ malE is 115%, and the yield per unit is 100% per unit yield of the parent strain. It was confirmed that both the glutamine production and the unit yield increased to 104% when as (Fig. 4a).

Example 5: glutamine Production strain Lesson 365 malE gene Fruiting bacteria Evaluation of glutamine production (fermentation tank)

Glutamine-producing strain 365 and malE in a 5 L fermenter consisting of the medium composition of Table 9 The glutamine production of gene deletion strains was compared.

As for the injection medium, A, B, C and D were sterilized separately, and 300 ml of seed culture was inoculated as a culture condition, and the injection liquid was performed at 1.85 L, a temperature of 30°C, agitation speed of 500 rpm, and aeration amount. It was incubated under the physical condition of 1.0 vvm. The 365-malE obtained in the present invention was cultured under the same conditions as the parent strain. Table 10 below relates to a comparison of the amount of glutamine produced (5 LJ/F) of the parent strain and the malE deletion strain.

As shown in the results of Table 10, the 365-Δ malE strain was found to have increased glutamine production without lowering the growth compared to the parent strain, resulting in a higher yield per unit. Both the glutamine and the glucose yield were relative values. The growth time was shortened by 15 hours compared to the parent strain until all of the sugar was consumed. As a result of culturing 365-Δ malE , an OD value of 83.3 was shown, showing a similar growth degree to that of the parent strain. In addition, 365-Δ glutamine production in malE is 103% when the production of the parental strain of 100%, per yields also a 365-Δ malE by the invention is 120% when the per yield of parent strain with 100% It was confirmed that both the glutamine production and the unit yield were increased (FIG. 4b).

Example 6: Arginine Production strain AR and ldhA gene Fruiting bacteria Arginine production evaluation (flask)

LdhA obtained in Example 1 above In order to confirm the amount of arginine production of the gene-deleting strain, the amount of arginine production of the parent strain, the arginine-producing strain AR, and the strain in which the ldhA gene was deleted, AR- ldhA , was compared. The two strains were compared with arginine production in a flask titer experiment in a medium consisting of the composition of Table 2 of Example 2.

Physical culture conditions were also the same as in Example 2. Table 11 below relates to a comparison of the amount of arginine produced by the parent strain and the ldhA deletion strain (flask titer experiment).

As shown in Table 11, the AR-Δ ldhA strain was found to have higher arginine production and yield compared to the parent strain. When comparing the arginine production of AR and AR-Δ ldhA cultivation results, when the production of the parent strain was 100%, the production of AR-Δ ldhA increased to 108%, and the yield per unit increased to 100% of the parent strain. When it was confirmed that it increased to 109% (Fig. 5a).

Example 7: Arginine Production strain AR and ldhA gene Fruiting bacteria Arginine production evaluation (fermentation tank)

Arginine-producing strains AR and ldhA The amount of arginine production was compared in a 5 L fermenter composed of the medium composition of Table 4 of Example 3 for the gene deletion strain.

Media conditions and physical culture conditions were also the same as in Example 3. Table 12 below relates to a comparison of the amount of arginine production (5 LJ/F) of the parent strain and the ldhA deletion strain.

As shown in Table 12, the AR- ldhA strain increased the production of arginine without lowering the growth compared to the parent strain, thereby increasing the yield per unit. As a result of culturing AR-Δ ldhA at the same time as the parent strain (84-86 hr), the result showed an OD value of 57.6, showing a similar growth degree to that of the parent strain. Further, the AR-Δ ldhA arginine yield is 110% when the production of the parental strain of 100%, per the yield also AR-Δ ldhA by the present invention as 110% when the per yield of parent strain to 100% of the It was confirmed that both the arginine production and the unit yield increased (FIG. 5B).

Example 8: Arginine Production strain AR and ldhA gene Fruiting bacteria Lactic acid production comparison (flask)

After culturing AR and AR-ΔldhA in the flask as in Example 6, the amount of lactic acid secreted from the culture supernatant to the outside of cells was compared.

As can be seen in Table 13, in the case of the ldhA- deficient strain, it was confirmed that the amount of lactic acid discharged to the outside of the cell was 0.3%, which was reduced by 28 times compared to 0.86% in the case of the parent strain. It could be confirmed that it decreased (FIG. 6). As described above, the decrease in lactic acid production increases the carbon flow from the pyruvic acid to the TCA ring, and it can be predicted that the productivity of arginine or glutamine is finally increased.

Example 9: glutamine Production strain 365 and ldhA gene Fruiting bacteria Evaluation of glutamine production (flask)

In order to confirm the glutamine production amount of the ldhA gene deletion strain obtained in Example 1, the glutamine production amount of the parent strain, glutamine-producing strain 365, and the strain in which the ldhA gene was deleted, 365- ldhA , was compared. The two strains were compared for glutamine production in a flask titer experiment in a medium consisting of the composition of Table 7 in Example 4.

Physical culture conditions were also the same as in Example 4. Table 14 below shows the parent strain and ldhA It relates to the comparison of the amount of glutamine produced by the deletion strain (flask titer experiment).

As can be seen in Table 14, the 365-Δ ldhA strain was found to have increased glutamine production compared to the parent strain, resulting in an increased yield. When comparing the glutamine production results of 365 and 365-Δ ldhA strain cultivation as a relative value, when the production of the parent strain is 100%, the production of 365-Δ malE is 112%, and the yield per unit is 100 per unit yield of the parent strain. When it was set as %, it was confirmed that both the glutamine production amount and the unit yield increased to 114% (FIG. 7a).

Example 10: glutamine Production strain Lesson 365 ldhA gene Fruiting bacteria Evaluation of glutamine production (fermentation tank)

Glutamine production strain 365 and ldhA gene deletion strain 365- ldhA were compared in a 5 L fermentor consisting of the medium composition of Table 9 in Example 5. Culture conditions and physical culture conditions were also the same as in Example 5.

The 365- ldhA obtained in the present invention was cultured under the same conditions as the parent strain. Table 15 below relates to a comparison of the amount of glutamine produced by the parent strain and the ldhA deletion strain (5 LJ/F).

As can be seen in Table 15, the 365-Δ ldhA strain was found to have increased glutamine production without lowering the growth compared to the parent strain, resulting in an increased yield. Both the glutamine and the glucose yield were expressed as relative values. As a result of culturing 365-Δ ldhA , it showed an OD value of 82.5, showing a degree of growth similar to that of the parent strain. In addition, 365-Δ glutamine production in ldhA is 108% when the production of the parental strain of 100%, per yields also a 365-Δ ldhA by the present invention as 115% when the per yield of parent strain with 100% It was confirmed that both the glutamine production and the unit yield were increased (FIG. 7b).

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, it is clear that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

<110> DAESANG CORPORATION <120> Mutant Strain with improved Amino acid production by inactivating malic enzyme or lactate dehydrogenase <130> PN130692 <160> 4 <170> KopatentIn 2.0 <210> 1 <211> 1179 <212> DNA <213> Corynebacterium glutamicum AR malE <400> 1 atgaccatcg acctgcagcg ttccacccaa aacctcaccc atgaggaaat cttcgaggca 60 cacgagggcg gaaagctctc cattagttcc actcgtccgc tccgcgacat gcgcgatctt 120 tcccttgctt acacccctgg tgttgctcag gtttgtgaag caatcaagga agatccagag 180 gttgcgcgca cccacacggg cattggaaac accgtcgcgg ttatttccga cggcaccgct 240 gttcttggcc ttggcgatat cggacctcag gcctcccttc ccgtcatgga gggcaaggct 300 cagctgttta gctctttcgc tggcctgaag gctatcccta tcgttttgga cgttcacgat 360 gttgacgctt tggttgagac catcgcagcc atcgcgcctt ctttcggtgc tatcaacttg 420 gaggacatct ccgctcctcg ttgcttcgag gtggagcgcc gcctcatcga gcgtctcgat 480 attccagtta tgcacgatga ccagcacggc accgctgtgg ttatcctcgc tgcgctgcgc 540 aactccctga agctgctgga tcgcaagatc gaagacctca agattgttat ttccggcgca 600 ggcgcagcgg gcgttgcagc tgtagatatg ctgaccaacg ctggagcaac cgatattgtg 660 gttcttgatt cccgaggcat catccacgac agccgtgagg atctttcccc agttaaggct 720 gctctcgcgg agaagaccaa ccctcgtggc atcagcggtg gcatcaatga ggctttcacc 780 ggcgcggacc tgttcattgg cgtgtccggc ggcaacatcg gcgaggacgc cctcaagctc 840 atggcaccac agccaatcct gttcaccctg gcgaacccaa ccccagagat cgatcctgag 900 ctgtctcaga agtacggcgc catcgtcgcg accggccgct ctgacctgcc taaccagatc 960 aacaacgtgc tcgcgttccc aggaattttc gccggcgctc tcgcagccaa ggctaagaag 1020 atcacccctg agatgaagct cgccgctgca gaggcaatcg ccgacatcgc agctgaggac 1080 ctcgaggtcg gccgcatcgt acctaccgcc ttggatcccc gcgtcgcccc agcagtcaag 1140 gcagctgtcc aggccgtcgc cgaagcgcaa aacgcttaa 1179 <210> 2 <211> 1179 <212> DNA <213> Corynebacterium glutamicum 365 malE <400> 2 atgaccacta gcttgcaacg ttccacccag aacctcaccc aggaagaaat ctttgaagca 60 cacgagggcg gaaagctctc tattagctcc acccgaccac ttcgcgatat gcgcgatctc 120 tccctggctt acacccccgg cgtagctaag gtatgcgagg caatcgcaga agatcctgag 180 gttgctcgca cccatacagg cattggcaac accgttgcag ttatttctga tggcaccgct 240 gttctgggct tgggcgatat cggaccacgc gcatcccttc cagttatgga aggaaaagct 300 cagctcttta gctcttttgc tggactgaag gccatcccta tcgttttgga tgtcaccgac 360 gttgacgctc tcgtagagac catcgcagcg atcgcacctt cctttggcgc tatcaacctg 420 gaagatatct ccgcgccacg ttgcttcgaa gtagagcagc gactcatcga gcgcctcgac 480 attcctgtca tgcacgatga tcagcatggc accgctgttg ttatcctcgc tgcattgaac 540 aactccctca agctgcttga ccgcaagatc gaagacctga agatcgtcat ctccggtgct 600 ggtgcagcag gtgttgcagc ggtagatatc ctcaccaacg caggcgcaac cgacatcgtt 660 gtcctcgatt cccgtggcat cctccacgac agccgcgaag acctctcccc agtgaaggcc 720 gatctggctg ctcgtaccaa cccacgcggc atcagcggtg gcatcaacga agcattcacc 780 gatgccaacc ttttcatcgg tgtttccggc ggaaacattg gtgaagatgc ccttaagttg 840 atggcaccac agccaatcct gttcaccctg gctaacccca ccccagagat cgatcccgag 900 ctgtccaaga agtacggcgc catcgttgcc accggacgct ccgatcttcc taaccagatc 960 aacaatgtat tggctttccc aggcatcttc gcaggtgctc tcgctgccaa ggcaaagaag 1020 atcaccccag agatgaagct tgctgctgca gcagccattg ctgacatcgc tgccgaagag 1080 ctcgaagttg gtcgcatcgt tccaaccgct ctggatccac gtgttgcccc tgcagtcaag 1140 gcagctgttc aggcagtcgc tgaggaacaa ggcgcataa 1179 <210> 3 <211> 957 <212> DNA <213> Corynebacterium glutamicum AR ldhA <400> 3 gtgggatcga aaatgaaaga aaccgtcggt aacaagattg tcctcattgg cgcaggagat 60 gttggagttg catacgcata cgcactgatc aaccagggca tggcagatca ccttgcgatc 120 atcgacatcg atgaaaagaa actcgaaggc aacgtcatgg acttaaacca tggtgttgtg 180 tgggccgatt cccgcacccg cgtcaccaag ggcacctacg ctgactgcga agacgcagcc 240 atggttgtca tttgtgccgg cgcagcccaa aagccaggcg agacccgcct ccagctggtg 300 gacaaaaacg tcaagattat gaaatccatc gtcggcgatg tcatggacag cggattcgac 360 ggcatcttcc tcgtggcgtc caacccagtg gatatcctga cctacgcagt gtggaaattc 420 tccggcttgg aatggaaccg cgtgatcggc tccggaactg tcctggactc cgctcgattc 480 cgctacatgc tgggcgaact ctacgaagtg gcaccaagct ccgtccacgc ctacatcatc 540 ggcgaacacg gcgacactga acttccagtc ctgtcctccg cgaccatcgc aggcgtatcg 600 cttagccgaa tgctggacaa agacccagag cttgagggcc gtctagagaa aattttcgaa 660 gacacccgcg acgctgccta tcacattatc gacgccaagg gctccacttc ctacggcatc 720 ggcatgggtc ttgctcgcat cacccgcgca atcctgcaga accaagacgt tgcagtccca 780 gtctctgcac tgctccacgg tgaatacggt gaggaagaca tctacatcgg caccccagct 840 gtggtgaacc gccgaggcat ccgccgcgtt gtcgaactag aaatcaccga ccacgagatg 900 gaacgcttca agcattccgc aaataccctg cgcgaaattc agaagcagtt cttctaa 957 <210> 4 <211> 945 <212> DNA <213> Corynebacterium glutamicum 365 ldhA <400> 4 atgaaagaaa ccgtcggaaa caaagtagtt cttattggtg caggtgatgt cggagtggct 60 tatgcttacg ccctcatcaa ccaaggaacg tgtgatcatc tcgccatcat tgacattgat 120 gaaaagaagc tcgaaggcaa tgtcatggat cttaaccatg gagttgtctg ggcagattcc 180 agaacacgcg ttagcaaagg cacctacgcc gattgtgaag acgcagcaat cgtagtgatt 240 tgcgccggag ccgcacaaaa gcccggtgaa acccgcctgc aattggtgga caaaaacgtc 300 aaaatcatga agaacatcgt cggcgatgtc atggacagtg gcttcgacgg catctttctg 360 gtggctagta atccagtgga catcctcacc tacgcagtat ggaaattctc cggcctcgat 420 tggcaccgtg ttatcggatc cggaacagtt ctggattcag cccgtttccg ctacatgctc 480 ggtgaactct atgaagtagc tccaagttct gtgcacgctt atatcatcgg cgaacacggc 540 gacactgagc ttcctgtgct gtcctcagcc accatcgcag gtgtgtccct cagccgcatg 600 ctggataaag accccagctt ggaagctcga ttggagaaaa tcttcgagga caccagagac 660 gccgcctatc acatcatcga tgccaaggga tcgacttctt atggcattgg catggggctt 720 gcgcgcatta cccgtgcggt tttgcaaaac caagatgtgg ctctgcctgt ctctgcctta 780 ttgcagggtg aatacggtga agaggatatc tacatcggca ctccagccat tgtgaatcgt 840 cgtggcattg cccgtgtcgt tgaactggaa attaatgatc atgagatgga aagattccaa 900 cattcggctg caaccttgaa ggaaatccaa caggaatttt tctag 945

Claims (8)

An arginine-producing mutant strain of Corynebacterium glutamicum comprising an inactivated malic enzyme, wherein the arginine producing mutant strain comprising the inactivated malate enzyme comprises wild-type or activated malate enzyme Mutant strains with 3-30% increase in the production of arginine compared to arginine-producing strains.
The method according to claim 1, wherein the inactivation of the malate enzyme is performed using malE Wherein the nucleotide sequence is inactivated by substitution, insertion, deletion, or a combination of nucleotide sequences encoding the gene.
delete delete delete delete And inactivating the malic acid enzyme to produce an arginine-producing Corynebacterium glutamicum mutant strain.
A method for producing arginine comprising culturing the mutant strain of claim 1 or 2.

Images