KR20130083690A - Microorganism producing l-isoleucine and process for preparing l-isoleucine using the same - Google Patents

Abstract

PURPOSE: A Corynebacterium glutamicum mutant strain is provided to supply enough L-threonine without the suppression of L-isoleucine feedback, thereby producing L-isoleucine with high efficiency and high yield. CONSTITUTION: A Corynebacterium glutamicum mutant strain (KCCM11248P) produces L-isoleucine. The mutant strain has resistance to L-isoleucine, L-threonine, and derivatives thereof. The L-isoleucine derivative is 4-thiaisoleucine (thiaile) or isoleucine-hydroxamate (ileHx). The L-threonine derivative is alpha-amino-beta-hydroxynorvaline (AHV). A method for producing L-isoleucine comprises the steps of: culturing the mutant strain (KCCM11248P); and collecting L-isoleucine from a culture of the mutant strain. [Reference numerals] (AA) L-leucine; (BB) Aminotransferase (ilvE); (CC) Ketoisocaproic acid; (DD) Isopropylmalic acid dehydrogenase (leuB); (EE) 3-isopropylmalic acid; (FF) Isopropylmalic acid dehydratase (leuD); (GG) 2-isopropylmalic acid; (HH) Isopropylmalic acid synthase (leuA); (II) L-valine; (JJ) Ketoisovaleric acid; (KK) Dihydroxyisovaleric acid; (LL) Acetolactic acid; (MM,NN,OO) Pyruvic acid; (PP) Acetohydroxyacid synthase (ilvBN); (QQ) Acetohydroxyacid isomer reductase (ilvC); (RR) Dihydroxy acid dehydratase (ilvD); (SS) Aminotransferase (ilvE); (TT) L-threonine; (UU) Threonine dehydratase (ilvA); (VV) 2-ketobutyric acid; (WW) Acetohydroxy butyric acid; (XX) Dihydroxy methyl valeric acid; (YY) Ketomethyl valeric acid; (ZZ) L-isoleucine

Description

Microorganism producing L-isoleucine and process for preparing L-isoleucine using the same

The present invention relates to a microorganism having improved L-isoleucine production capacity and a method for producing L-isoleucine using the same.

L-amino acids are widely used in functional food additives, animal nutrition, and the pharmaceutical industry as the basic units of protein. Branched-chain amino acid out of a total of 20 amino acids refers to the three kinds of L-valine, L- leucine, L-isoleucine, and is increasing in industrial value. Branched chain amino acids play an important role in maintaining and forming skeletal livers in the human body, and have been reported to be functional in insulin regulation and muscle maintenance and gain (Andrea tom et al, (2006) The journal of nutrition, 136 , 324s-330s). In particular, L-isoleucine is metabolized in the muscle to produce energy, and is involved in the production of hemoglobin to reduce fatigue and promote growth. Accordingly, it is widely used as a sap preparation and a nutritional supplement, and is increasingly used in sports nutrition.

Corynebacterium glutamicum is used as a representative microorganism for industrial production of L-isoleucine. The microorganism synthesizes L-isoleucine via three intermediate metabolites using pyruvate and 2-ketobutyrate as precursors (see FIG. 1). 2-aceto-2-hydroxybutyrate is synthesized from the two precursors, and 2,3-dihydroxy-3-methylvalerate (2,3-dihydroxy-3-methylvalerate ) And 2-keto-3-methylvalerate to finally produce L-isoleucine. Acetohydroxy acid synthase, acetohydroxy acid isomeroreductase, dihydroxy acid dehydratase, and An enzyme called aminotransferase is used (Jin hwan park et al, Appl microbial biotechnol, (2010) 85: 491-506).

Acetohydroxy acid synthase, which is important in the biosynthesis of L-isoleucine in the Corynebacterium glutamicum strain, is encoded by the ilvBN gene, and is expressed by the final product L-isoleucine. It has a feedback inhibitory effect that inhibits the activity of the enzyme. In addition, threonine dehydratase, which produces 2-ketobutyrate, is also feedback-suppressed by L-isoleucine, thereby regulating the expression of genes involved in L-isoleucine biosynthesis and Modulating enzyme activity is known to be an important factor in producing high yield L-isoleucine producing strains (Jin hwan park et al, Biotechnology journal, (2010) 560-577). In addition, as shown in FIG. 1, since L-isoleucine, L-valine, and L-leucine are produced through the same biosynthetic pathway, in order to mass-produce L-isoleucine, a precursor of 2-ketobutyrate is used. A smooth supply of the L-threonine used should reduce production of other branched chain amino acids and allow for continued L-isoleucine synthesis. In order to solve this problem, there is a document that enhances the production capacity of L-threonine by using the α-amino-β-hydroxynorvaline (α-amino-β-hydroxynorvaline) ( Cayo ramos et al, Applied and environmental microbiology, (1992) 1677-1682), a method for imparting L-isoleucine production capacity to microorganisms having L-threonine production ability (Korean Patent Publication No. 2011-0058731), There are prior art patents such as microorganisms which simultaneously produce L-threonine and L-isoleucine (Korean Patent Publication No. 2002-0013777). In addition, the suppression of feedback of threonine dehydratase (4-Jisoleucine, John J. wasmuth, Journal of bacteriology, (1973) 562-570) was performed using 4-thiaisoleucine, an isoleucine derivative. It has been reported that the mutant strains resistant to the confirmed literature and isoleucine-hydroxamate improved L-isoleucine production ability (M. kisumi, Journal of general microbiology, (1971) 69 291-297). In order to improve the production method of L-Isoleucine, the method of developing AHAS which gave L-isoleucine producing strains to increase L-isoleucine production efficiency rather than L-valine production (Korean Publication No. 2011-0061780). Research has been published that improves yields by supplying oxygen or by changing physical conditions such as pH during fermentation (Zhihian peng et al, Bioprocess biosyst eng, (2010) 33: 339-345).

However, the L-isoleucine producing microorganisms studied so far have separate resistance to some substances of the L-isoleucine biosynthetic pathway, so that various substances related to feedback regulation of L-isoleucine biosynthesis are There remains a need for the development of L-isoleucine producing microorganisms that are resistant to.

Under these circumstances, the present inventors have made diligent efforts to develop L-isoleucine producing microorganisms with better yields than conventional strains. As a result, L-threonine derivative α-amino-β-hydroxynorvaline (α-amino -[beta] -hydroxynorvaline) and L-isoleucine derivatives 4-thiaisoleucine and isoleucine-hydroxamate confer common resistance, resulting in high yields The present invention was completed by confirming to produce L-isoleucine.

One object of the present invention is to provide a high yield of Corynebacterium glutamicum mutants for L-isoleucine production.

Another object of the present invention is to provide a method for producing L-isoleucine using the mutant strain.

It is still another object of the present invention to provide a method for preparing a high yield L-isoleucine mutant in Corynebacterium glutamicum.

As one embodiment for achieving the above object, the present invention is Corynebacterium glutamicum for producing L-isoleucine ( Corynebacterium) glutamicum ) provides a variant strain KCCM11248P.

As used herein, the term "L-isoleucine" is one of the essential amino acids structurally corresponding to the branched chain amino acids along with L- valine, L- leucine, HO ₂ CCH (NH ₂ ) CH (CH ₃ ) CH ₂ CH ₃ Phosphorus L-amino acid.

Meanwhile, in microorganisms, biosynthesis of L-isoleucine is synthesized through a four-step biosynthesis process using pyruvate and 2-ketobutyrate as precursors, as shown in FIG. 1. However, the biosynthesis steps are commonly used for the biosynthesis of the remaining branched chain amino acids, namely L-valine and L-leucine, and the fermentation is performed because a sufficient supply of L-threonine, a precursor necessary for biosynthesis of L-isoleucine, must also be achieved. There was a problem in mass production of L-isoleucine through. The mutant of the present invention has a resistance to the final product L-isoleucine or a derivative thereof to release the feedback inhibition, and has a resistance to the L-threonine derivative to release the feedback inhibition to provide sufficient precursor supply It is a novel microorganism with improved L-isoleucine production capacity.

The mutant of the present invention may preferably be resistant to L-isoleucine or derivatives thereof, L-threonine or derivatives thereof, and most preferably resistant to derivatives of L-isoleucine and L-threonine derivatives. Can have

As used herein, the term "derivative" refers to the inhibition of L-isoleucine or L-threonine from microorganisms by causing feedback inhibition in relation to the biosynthesis of L-isoleucine or the precursor L-threonine, the final product of the present invention. It can mean known compounds that can inhibit the production capacity, for example L-isoleucine derivatives are 4-thiaisoleucine (thiaile) or isoleucine-hydroxamate (isoleucine-hydroxamate, ileHx) And, L- threonine derivatives include α-amino-β-hydroxynorvaline (α-amino-β-hydroxynorvaline, AHV) and the like, but is not limited thereto. Preferably the mutant may be resistant to one or more substances selected from the group consisting of 4-thiaisoleucine, isoleucine-hydroxyxamate and α-amino-β-hydroxynorvaline, most preferably 4- It can be resistant to both thiisoleucine, isoleucine-hydroxyxamate and α-amino-β-hydroxynorvaline.

In general, it is known that L-isoleucine biosynthesis is inhibited when L-isoleucine accumulates at a certain concentration in cells. Therefore, since the strain resistant to the derivative is a strain having the ability to produce L-isoleucine even under conditions containing high concentration of L-isoleucine by inhibiting L-isoleucine, the present invention According to one embodiment of the present inventors selected a high concentration of L-isoleucine producer using the derivative. In addition, since L-threonine is used as a precursor of 2-ketobutyric acid for mass production of L-isoleucine, strains resistant to L-threonine are released from the inhibition of L-threonine. Since a sufficient supply of the precursor L- threonine is made, L-threonine derivatives were also used to select high concentrations of L-isoleucine producers.

According to the present invention, the microbial mutant strain with improved production capacity of L-isoleucine selects the desired mutant strain by mutating the parent strain. At this time, the mutagenesis of the microorganism may be carried out by various means well known in the art, and may use either a physical or chemical mutagenesis. For example, suitable chemical mutagens for the present invention include N-methyl-N'-nitro-N-nitrosoguanidine (NTG), diepoxybutane, ethylmethane Sulfonates, mustard compounds, hydrazine and nitrous acid, including but not limited to these compounds. In addition, physical mutagens may include, but are not limited to, ultraviolet and gamma radiation.

During mutagenesis, the parent strain is affected by mutagenesis at a concentration sufficient to leave a certain size of surviving population. The size varies with the type of mutagenesis and is dependent on the amount of mutation that the mutagenesis causes in the living population at a constant killing rate. For example, for NTG, the desired killing rate should leave approximately 10% to 50% of the starting population. Mutagenesis by nitrous acid should leave approximately 0.01% to 0.1% of the starting population and mutagenesis by ultraviolet light should leave approximately 1.0%. According to one embodiment of the present invention, NTG was used as a method for inducing mutations in the parent strain to produce microbial mutants with improved L-isoleucine production capacity.

In one embodiment of the present invention Corynebacterium glutamicum KFCC 11040 ( Corynebacterium glutamicum to produce glutamic acid in order to produce a mutant strain with improved production capacity of L-isoleucine KFCC 11040, Korean Patent Publication No. 2000-0002407) was used as a parent strain. After random mutation of the parent strain, L-isoleucine derivative 4-thiaisoleucine (thiaile), isoleucine-hydroxyxamate (ileHx) and L-threonine derivative α-amino-β-hydride Staining the medium with Loxynorvaline (AHV) added together, selected common resistance mutants with resistance at concentrations of 1 mM, 1 mg / ml and 25 mg / ml for each were named KCJI-38. In addition, the common resistance mutant strain was confirmed that the L- isoleucine production increased by more than 13 times compared to the parent strain (see Table 1). Corynebacterium glutamicum mutant ( Corynebacterium glutamicum ( KCJI-38) was deposited on January 9, 2012 with the Korea Microbiological Conservation Center, an international depository organization located at Yurim Building, 361-221, Hongje 1-dong, Seodaemun-gu, Seoul, Korea.

As another aspect, the present invention provides a method for producing L-isoleucine comprising the step of culturing the mutant strain.

Preferably, the method for preparing L-isoleucine may further include recovering L-isoleucine from the culture medium in which the mutant strain is cultured.

The term "cultivation" in the present invention means cultivation of microorganisms under moderately artificially controlled environmental conditions. In the present invention, the method of culturing Corynebacterium glutamicum mutants to produce L-isoleucine can be carried out using a method of culturing Corynebacterium glutamicum, which is well known in the art. Specifically, examples of the culture method include, but are not limited to, batch culture, continuous culture, and fed-batch culture. Various such methods are disclosed, for example, in “Biochemical Engineering” (James M. Lee, Prentice-Hall International Editions, (1991) pp138-176).

The medium used for the culture should meet the requirements of the particular strain in an appropriate manner. Culture media for Corynebacteria strains are known (eg, Manual of Methods for General Bacteriology. American Society for Bacteriology.Washington D.C., USA, 1981). Sugar sources that may be used include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, stearic acid Fatty acids such as linoleic acid, alcohols such as glycerol, ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture. Examples of nitrogen sources that may be used include peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. Nitrogen sources can also be used individually or as a mixture. Personnel that may be used may include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. In addition, the culture medium should contain metal salts such as magnesium sulfate or iron sulfate required for growth. In addition to these substances, essential growth substances such as amino acids and vitamins may be included. In addition, precursors suitable for the culture medium may be used. The above-mentioned raw materials can be added to the culture in a batch manner or in a continuous manner by an appropriate method.

Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or acid compounds such as phosphoric acid or sulfuric acid can be used in a suitable manner to adjust the pH of the culture. In addition, bubble formation can be suppressed by using a defoaming agent such as a fatty acid polyglycol ester. Inject oxygen or an oxygen-containing gas (eg air) into the culture to maintain aerobic conditions. The temperature of the culture is usually 20 ° C to 45 ° C. Incubation is continued until the desired amount of L-isoleucine production is achieved. Usually for 10 to 160 hours for this purpose. L-isoleucine may be excreted in the culture medium or contained in the cells.

L-isoleucine production method of the present invention comprises the step of recovering L-isoleucine from cells or culture. Methods for recovering L-isoleucine from cells or cultures may include, but are not limited to, methods known in the art, such as centrifugation, filtration, anion exchange chromatography, crystallization, and HPLC. According to one embodiment of the invention, the culture was centrifuged at low speed to remove the biomass and the obtained supernatant was separated by liquid high-speed chromatography.

As another aspect, the present invention comprises the step of selecting a variant strain resistant to L-isoleucine derivatives and L- threonine derivatives in Corynebacterium glutamicum, Coryne for L-isoleucine production Provided is a method for producing bacterium glutamicum mutant strain.

The parent strain Corynebacterium glutamicum may be a native or mutant strain.

Preferably, the mutation can be made by performing a mutation method.

L-isoleucine derivatives, L-threonine derivatives and mutation methods are as described above.

In the present invention, the method of preparing a mutant strain can select a higher yield of Corynebacterium glutamicum mutants than the existing strains by selecting mutants having resistance at the same time both L-isoleucine derivatives and L-threonine derivatives.

Corynebacterium glutamicum mutants of the present invention have a common resistance to L-isoleucine, L-threonine and derivatives thereof, without receiving feedback inhibition to L-isoleucine, and the precursor L-tsu Sufficient supply of leonin has been achieved to improve the production capacity of L-isoleucine. Therefore, according to the method for producing L-isoleucine of the present invention using the microorganism, L-isoleucine can be produced with high efficiency and high yield.

1 is a diagram showing the biosynthetic pathway of the same branched chain amino acid with the biosynthesis process of L-isoleucine, the final product of the present invention. As shown in FIG. 1, branched chain amino acids are produced through biosynthesis by the same enzyme.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example  One: Artificial variation  Variation selection through

In order to obtain a microbial mutant strain with improved production capacity of L-isoleucine, the microorganisms were induced using the following method.

Specifically, the parent strain Corynebacterium glutamicum KFCC 11040 ( Corynebacterium glutamicum KFCC 11040, Republic of Korea Patent Publication No. 2000-0002407) was incubated for 16 hours in an activated medium and incubated for 14 hours by inoculating the activated strain in a sterilized seed medium at 121 ℃ for 15 minutes, and then recovered 5 ml of the culture solution It was. The recovered culture was washed with 100 mM citric acid buffer, and then NTG (N-Methyl-N'-nitro-N-nitrosoguanidine) was added to a final concentration of 200 mg / l, followed by 20 minutes of treatment. And washed with 100 mM phosphate buffer. The mortality rate was 85% when NTG-treated strains were plated on the minimum medium.

The NTG-treated strains were converted to thiaile, ileHx to obtain common resistance mutants against 4-thiaisorusine (thiaile), isoleucine-hydroxyxamate (ileHx) and α-amino-β-hydroxynorvaline (AHV). , The final concentration of AHV 1 mM, 1 mg / ㎖, 25 mg / ㎖ was added to a minimum medium added to each, and cultured at 30 ℃ for 5 days, to obtain a common resistance mutant strain of thiaile, ileHx and AHV.

The mutant strain obtained by the above method was named Corynebacterium glutamicum KCJI-38 ( Corynebacterium glutamicum , KCJI-38), and was deposited with the Korea Microorganism Conservation Center on January 9, 2012 and was given accession number KCCM11248P.

The composition of the medium used in Examples 1 and 2 is as follows.

< Activation Badge >

Juicy 1%, Polypeptone 1%, Sodium Chloride 0.5%, Yeast Extract 1%, Agar 2%, pH 7.2

< Species >

Glucose 5%, bactopeptone 1%, sodium chloride 0.25%, yeast extract 1%, urea 0.4%, pH 7.2

< Minimum badge >

Glucose 1.0%, Ammonium Sulfate 0.4%, Magnesium Sulfate 0.04%, Potassium Phosphate 0.1%, Urea 0.1%, Thiamine 0.001%, Biotin 200 μg / L, Agar 2%, pH 7.2

Example  2: L- Isoleucine  Production variation mutant L- Isoleucine  Productivity Survey

The high concentration of thiaile, ileHx and AHV common resistance mutants obtained in Example 1 were cultured in the following manner to confirm the L-isoleucine productivity of Corynebacterium glutamicum KCJI-38 (KCCM11248P).

L-isoleucine was prepared by inoculating the mother strain and the mutant strain in a 250 ml corner-bottom flask containing 25 ml of the production medium, followed by shaking culture at 200 rpm for 30 hours at 30 ° C.

The composition of the production medium used in Example 2 is as follows.

< Production badge >

Glucose 10%, Yeast Extract 0.2%, Ammonium Sulfate 1.6%, Potassium Monophosphate 0.1%, Magnesium Sulfate 0.1%, Ferrous Sulfate 10 mg / L, Manganese Sulfate Monohydrate 10 mg / L, Biotin 200 ㎍ / ℓ, pH 7.2

After the end of the culture, L-isoleucine production was measured using high-performance liquid chromatography, and the L-isoleucine concentration in the culture solution for each strain tested was shown in Table 1 below.

Comparison of L-Isoleucine Productivity between Mother Line and KCJI-38 (KCCM11248P) Corynebacterium glutamicum
KFCC 11040 (parent strain) Corynebacterium glutamicum
KCJI-38 (variant strain) L-isoleucine concentration (g / l) 0.1 1.3

As a result, as shown in Table 1, the parent strain Corynebacterium glutamicum KFCC 11040 (Korean Patent Publication No. 2000-0002407) produced L-isoleucine at a concentration of 0.1 g / L, Variation strain Corynebacterium glutamicum KCJI-38 (KCCM11248P) according to the present invention produces L-isoleucine at a concentration of 1.3 g / L, which increases the productivity of L-isoleucine by at least about 13 times that of the parent strain. Confirmed.

The results show that the mutant strains resistant to L-isoleucine derivatives are not subject to feedback inhibition as a result, and that the mutant strains resistant to L-threonine derivatives can supply L-isoleucine with high efficiency. And high yield.

Korea Microorganism Conservation Center (overseas) KCCM11248P 20120109

Claims (8)

Corynebacterium glutamicum mutant KCCM11248P for L-isoleucine production.
The variant strain KCCM11248P according to claim 1, wherein said mutant strain is resistant to L-isoleucine, L-threonine, and derivatives thereof.
The method of claim 2, wherein the L-isoleucine derivative is 4-thiaisoleucine (thiaile) or isoleucine-hydroxamate (ileHx), and the L-threonine derivative is α- Variant KCCM11248P which is amino-β-hydroxynorvaline (AHV).
A method for producing L-isoleucine comprising culturing the variant strain KCCM11248P of any one of claims 1 to 3.
The method of claim 4, further comprising recovering L-isoleucine from the culture of the mutant strain KCCM11248P.
The method according to claim 4, wherein the incubation temperature is 20 to 45 ℃, incubated for 10 to 160 hours under aerobic conditions.
A method of preparing corynebacterium glutamicum mutants for L-isoleucine production, comprising the step of selecting mutants having resistance to L-isoleucine derivatives and L-threonine derivatives in Corynebacterium glutamicum.
The method of claim 7, wherein the variant strain selection is by a mutation method.

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