KR101233666B1 - Production of Isoleucine Using Mutant Bacteria - Google Patents

Abstract

The present invention provides a bacterial variant strain with increased isoleucine production capacity and a method for producing the same. The present invention is a strain capable of increasing the production capacity of isoleucine and at the same time effectively reducing the production of valine by inactivating the nucleotide encoding the ilvB gene in bacteria having isoleucine production capacity. The present invention shows a low production of valine while maintaining the production capacity of isoleucine even when cultured at a temperature higher than the conventional culture temperature, such as cooling water that is added during bacterial culture when producing isoleucine using the strain of the present invention. It has the advantage of saving raw material costs. In addition, the present invention can not only purify isoleucine with high purity by reducing the production capacity of valine, a by-product of bacteria, but also improve the recovery rate of isoleucine, thereby significantly lowering the production cost of illleucine.

Description

Production of Isoleucine Using Bacterial Mutant Strains {Production of Isoleucine Using Mutant Bacteria}

The present invention relates to bacterial strains with increased isoleucine production capacity.

The amino acid L-isoleucine is a commercially meaningful product used in animal feed, human food additives and the pharmaceutical field. For this reason, it is important in the industrial sense to improve the production of L-isoleucine.

L-isoleucine shares a major biosynthetic pathway with the branched chain amino acids L-valine, L-leucine. In particular, because L-isoleucine and L-valine are very similar in chemical structure, isoelectric point, and solubility, so many process steps and costs are required to produce high purity single amino acid to L-isoleucine. Therefore, it is difficult to recover with high yield. In order to reduce the production cost of isoleucine, it is important to develop strains with low content of valine, which is a major by-product produced with isoleucine.

There have been several methods from the prior art for developing L-isoleucine producing strains. The most common amino acid production strain development methods include resistance strain screening for intermediate metabolites or end products using mutagenesis, and deletion of active genes and / or enhancement of target product production control genes through chromosomal mutation. In other words, mutagenesis may be used to select mutant strains resistant to similar structures of threonine and isoleucine, which are the main intermediate metabolites of isoleucine biosynthesis.

However, if the above-mentioned general amino acid strain development method such as the above-mentioned mutagenesis is applied to isoleucine-producing strains, it shares the main biosynthetic process with isoleucine, and can also affect the production of valine, a by-product discharged from the cell in a similar manner. There is a possibility.

Therefore, in order to reduce the production cost by increasing the recovery yield of isoleucine, there is a need for a new strain development method for developing a strain having both isoleucine production capacity and a low content of valine, which is a major by-product.

The present inventors earnestly tried to develop bacterial strains that can efficiently produce isoleucine. As a result, the present invention was completed by identifying that isoleucine producing ability can be increased by inactivating acetohydroxyacid synthase I of isoleucine producing ability bacterial strain.

Therefore, an object of the present invention is to provide a bacterial variant strain with increased isoleucine production capacity.

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

According to one aspect of the present invention, the present invention provides a bacterial variant with increased isoleucine production capacity including inactivated acetohydroxyacid synthase I.

The present inventors made intensive efforts to develop a bacterial strain capable of efficiently producing isoleucine, and as a result, isoleucine production by inactivating acetohydroxyacid synthase I of isoleucine producing bacterial strains It was found that the capacity could be increased.

The present invention is commonly involved in the biosynthesis process of isoleucine (L-Isoleucine; Ile), valine (L-Valine; Val), and leucine (L-leucine) of isoleucine-producing bacterial strains using genetic recombination techniques. A nucleotide encoding the ilvB gene, which is a large subunit of aHAS I (acetohydroxyacid synthase I), is an inactivated strain.

According to a preferred embodiment of the present invention, the present invention is a strain prepared by substitution, insertion, deletion or a combination of nucleotide sequences of ilvB .

The present invention is a bacterial mutant strain that increases the production of isoleucine and at the same time significantly reduces the production of by-product valine, and increases the recovery rate of isoleucine and isoleucine in separating and purifying isoleucine from bacteria and / or bacterial culture. Is a bacterial variant that effectively reduces the production of valine, which is a barrier in the isolation and purification of

The expression “decrease in valine production capacity” used while expressing bacterial variant strains in the context of the present invention means a reduced amount of valine produced from wild-type bacterial strains available from nature.

According to a preferred embodiment of the present invention, the present invention is a strain having reduced production of valine compared to wild type bacterial strains.

According to a preferred embodiment of the present invention, the present invention is a strain in which the ratio of valine yield to isoleucine yield is 1-10%.

The expression “proportion of valine yield” used in expressing bacterial variant strains in the present specification means a ratio indicating the amount of valine produced in percentage based on the unit weight or volume of isoleucine produced by the bacteria. For example, if the amount of isoleucine produced by bacteria is 100 g and the amount of valine is 10 g, the ratio of valine production to isoleucine production is 10%.

The strain used in the present invention may be used without limitation as long as it is a bacterial strain having an isoleucine producing ability in nature.

According to a preferred embodiment of the present invention, the strain in the present invention is Escherichia sp.) strain, more preferably Escherichia coli ), Escherichia albertii ), Escherichia blattae ), Escherichia fergusonii ) ( Escherichia hermannii ) or Escherichia vulneris , even more preferably Escherichia coli or Escherichia blotae , most preferably Escherichia coli.

The present invention provides acetohydroxy acid by disabling the enzymatic activity of ilvB in acetohydroxy acid thinase I ( ilvBN ), acetohydroxy acid thinase II ( ilvGM ) and acetohydroxy acid thinase III ( ilvHI ). Relatively low pyruvate affinity for thinase enzyme activity reduces the production of L-Valine (Val), acetohydroxy acid thinase II ( ilvGM ) and acetohydroxy acid thin It is a bacterial variant strain that maintains and increases the production capacity of isoleucine by maintaining the activity of Saase III ( ilvHI ).

According to a preferred embodiment of the invention, the invention is a strain comprising activated acetohydroxy acid thinase II and acetohydroxy acid thinase III.

The present invention can be cultured through a known method of culturing bacterial strains. The medium may be natural or synthetic medium. As the carbon source of the medium, for example, glucose, sucrose, dextrin, glycerol, starch and the like can be used, and as the nitrogen source, peptone, meat extract, yeast extract, dried yeast, soy cake, urea, thiourea, ammonium salt, knight Rates and other organic or inorganic nitrogen-containing compounds may be used, but are not limited to these components.

Examples of inorganic salts contained in the medium include, but are not limited to, phosphates such as magnesium, manganese, potassium, calcium and iron, nitrates, carbonates, chlorides and the like.

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

According to another aspect of the present invention, the present invention provides a method for producing bacterial strains having increased isoleucine production capacity comprising inactivating acetohydroxyacid synthase I.

Inactivating acetohydroxyacid synthase I in the present invention may be carried out through known methods.

In the method of the present invention, the step of inactivating acetohydroxy acid thinase I is performed by CaCl 2 method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973)). , Hanahan, D., J. Mol. Biol., 166: 557-580 (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973); 1983) and electroporation methods (Dower, WJ et al., Nucleic. Acids Res., 16: 6127-6145 (1988)) and the like.

The present invention also includes the steps of isolating and obtaining a transformed bacterial strain from a non-transformed bacterial strain.

In the present invention, a labeled vector can be used in the step of separating and obtaining the transformed bacterial strain from the non-transformed bacterial strain.

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

Selective markers used in the present invention include antibiotic resistance genes commonly used in the art and include, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline. Resistance genes, and preferably ampicillin or kanamycin resistance genes in view of cost.

Since the present invention is a method for producing the same strain as the bacterial strain with increased isoleucine production capacity, including the inactivated acetohydroxyacid synthase I (acetohydroxyacid synthase I), the common content between the two In order to avoid excessive complexity, the description is omitted.

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

(Iii) The present invention provides a bacterial variant strain with increased isoleucine production capacity and a method for producing the same.

( Ii ) The present invention is a strain capable of increasing the production capacity of isoleucine and effectively reducing the production of valine by inactivating the nucleotide encoding the ilvB gene in bacteria having isoleucine production capacity.

(Iii) The present invention shows a low valine production amount while maintaining the production of isoleucine even when cultured at a temperature higher than the existing culture temperature, so that when the production of isoleucine using the strain of the present invention is added during bacterial culture It has the advantage of saving raw material costs such as cooling water.

(Iii) In addition, the present invention can not only purify isoleucine with high purity by reducing the production of valine, a by-product of bacteria, but also improve the recovery rate of isoleucine, thereby greatly lowering the production cost of irrucine. .

1 is a gel photograph showing the results of PCR analysis confirming the deletion of the ilvB gene.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Throughout this specification, "%" used to denote the concentration of a particular substance is intended to include solids / solids (wt / wt), solid / liquid (wt / The liquid / liquid is (vol / vol)%.

Example  One: ilvB Production of a mutant strain in which a gene is deleted

The ilvB gene was deleted by one step inactivation method (Warner et al., PNAS, 6: 6640-6645 (2000)) and the antibiotic resistance endowment marker gene was removed. The nucleotide sequence of the ilvB gene to be deleted is the same as in SEQ ID NO: 1.

Primer pairs in Table 1 and pKD13 plasmid (GenBank accession number AY048744) to prepare ilvB deletion strains, which are genes encoding the large subunit of acetohydroxyacid synthase I (AHAS I). ) PCR reaction (total reaction volume is 50 μl, after 1 minute 1 cycle at 94 ° C., 30 seconds at 94 ° C., 30 seconds at 55 ° C. and 1 minute 30 seconds at 72 ° C., then 72 ° C.) 2 min and 5 min at 10 ° C.), and the resulting DNA fragment was electrophoresed into Escherichia coli DS44 (Escherichia coli, DS44; KCTC 11602BP) cells containing pKD46 (GeneBank No.AY048746). One step inactivation; Warner et al., PNAS, 6: 6640 (2000). Thereafter, PCR reaction was performed on the cell line showing kanamycin resistance to confirm the deletion of the ilvB gene. In the PCR reaction, the PCR reaction was performed using the ilvB _CF and K primers of Table 1 in the same manner as described above. Since k primer acts on the fragmentation DNA inserted into the chromosome, DS44Δ ilvB containing the fragmentation DNA was formed in a size of about 1.6 kb as expected, and in the case of DS44, the PCR reaction did not occur because the fragmentation DNA was not present. Not formed (see FIG. 1). The process of removing the antibiotic resistance marker gene was performed using the strain in which the ilvB gene deletion was confirmed. In the process, pLP20 plasmid was introduced into the ilvB gene deletion strain to induce FLP recombination. Then, the antibiotic resistance marker gene was removed by culturing the ilvB gene deletion strain in the LB plate without or without antibiotics.

primer Base sequence ilvB_F 5'-GTTTAAGCACCTCCCGGAAAGTCGGCCCAGAAGAAAAGGACTGGAGC
ATGATTCCGGGGATCCGTCGACC-3 ' ilvB_R 5'- ACGTTGTCATGAGTTGTGTTTTGCATGGCTTATTCCCCCA CCATTTCAGTTGTA
GGCTGGAGCTGCTTCG-3 ' ilvB_CF 5'-cgcaaactgtgatcgatac-3 ' K 5'-cggccacagtcgatgaatcc-3 '

Example  2. Escherichia Collai DS44  Strains and ilvB  Gene deletion strains Isoleucine  And valine yield assessment

In order to confirm the production amount of isoleucine and valine of the ilvB gene deletion strain obtained in Example 1, Escherichia coli DS44 (Esherichia, a parent strain) coli , DS44; KCTC 11602BP) and the production of isoleucine and valine of DS44ΔilvB, a strain lacking the ilvB gene, were compared. The two strains were compared with the production amount of isoleucine and valine in a 5 L fermenter consisting of F-1 medium composition of Table 2.

ingredient Injection medium concentration Additional medium concentration glucose 80 g / ℓ 550 g / ℓ Corn Soak 20 g / ℓ - Ammonium Sulfate 20 g / ℓ 1 g / ℓ Phosphoric Acid 15 g / ℓ 1 g / ℓ Fumaric acid 1 g / ℓ - Sodium glutamate 7 g / ℓ - Sodium citrate 1 g / ℓ - Choline-HCl 1 g / ℓ - Thiamine-HCl 5 mg / l - Pyridoxine-HCl 10 mg / l - Nicotinic acid 5 mg / l - Biotin 5 mg / l - Calcium chloride 5 mg / l - Cobalt chloride 5 mg / l - Iron sulfate 20 mg / l - Manganese sulfate 5 mg / l - Zinc sulfate 5 mg / l - Copper sulfate 5 mg / l - Sodium hydroxide 10 g / ℓ -

The culture medium was carried out with 20 L of the injected liquid and 342 mL of the additional liquid. The physical culture conditions were DS44 (KTCK 11602BP) incubated under a temperature of 27 ° C., agitation speed of 500 rpm, and aeration rate of 1 vvm. DS44ΔilvB obtained in the present invention was a temperature condition of 30 ℃ higher than the culture conditions of DS44 3 ℃, other stirring rate and aeration portion was incubated under the same conditions.

In the fermentation culture using microorganisms, if the culture temperature is increased, the growth rate is increased. The same applies to fermentation cultures for isoleucine production. That is, in fermentation culture for isoleucine production, increasing the culture temperature increases the intracellular pyruvate concentration, which induces the production of valine. Therefore, DS44ΔilvB obtained in the present invention was carried out by raising the culture temperature higher than the parent strain DS44, and as a result, it was found that the V / I ratio was reduced even at a high temperature as shown in Table 3. Comparison of Isoleucine and Valine Production in Probiotics and ilvB Deficient Microorganisms (5L J / F)

Strain name Incubation time (hr) Isoleucine (%) Valine (%) V / I ratio (%) DS44 75 2.05 0.29 14 DS44ΔilvB 53 2.84 0.17 6

As shown in Table 3, the DS44ΔilvB strain maintained or increased isoleucine production ability, and valine production capacity was reduced in the conditions known to generate a large amount of valine. Comparing the V / I ratio of the DS44 and DS44ΔilvB culture results, the V / I ratio of DS44ΔilvB to the parent strain was 14% to 6%, and the V / I ratio was reduced by 57% based on the 14% V / I ratio according to the present invention. I could confirm that

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

<110> Daesang Corporation <120> Production of Isoleucine Using Mutant Bacteria <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 1689 <212> DNA <213> Escherichia coli <400> 1 atggcaagtt cgggcacaac atcgacgcgt aagcgcttta ccggcgcaga atttatcgtt 60 catttcctgg aacagcaggg cattaagatt gtgacaggca ttccgggcgg ttctatcctg 120 cctgtttacg atgccttaag ccaaagcacg caaatccgcc atattctggc ccgtcatgaa 180 cagggcgcgg gctttatcgc tcagggaatg gcgcgcaccg acggtaaacc ggcggtctgt 240 atggcctgta gcggaccggg tgcgactaac ctggtgaccg ccattgccga tgcgcggctg 300 gactccatcc cgctgatttg catcactggt caggttcccg cctcgatgat cggcaccgac 360 gccttccagg aagtggacac ctacggcatc tctatcccca tcaccaaaca caactatctg 420 gtcagacata tcgaagaact cccgcaggtc atgagcgatg ccttccgcat tgcgcaatca 480 ggccgcccag gcccggtgtg gatagacatt cctaaggatg tgcaaacggc agtttttgag 540 attgaaacac agcccgctat ggcagaaaaa gccgccgccc ccgcctttag cgaagaaagc 600 attcgtgacg cagcggcgat gattaacgct gccaaacgcc cggtgcttta tctgggcggc 660 ggtgtgatca atgcgcccgc acgggtgcgt gaactggcgg agaaagcgca actgcctacc 720 accatgactt taatggcgct gggcatgttg ccaaaagcgc atccgttgtc gctgggtatg 780 ctggggatgc acggcgtgcg cagcaccaac tatattttgc aggaggcgga tttgttgata 840 gtgctcggtg cgcgttttga tgaccgggcg attggcaaaa ccgagcagtt ctgtccgaat 900 gccaaaatca ttcatgtcga tatcgaccgt gcagagctgg gtaaaatcaa gcagccgcac 960 gtggcgattc aggcggatgt tgatgacgtg ctggcgcagt tgatcccgct ggtggaagcg 1020 caaccgcgtg cagagtggca ccagttggta gcggatttgc agcgtgagtt tccgtgtcca 1080 atcccgaaag cgtgcgatcc gttaagccat tacggcctga tcaacgccgt tgccgcctgt 1140 gtcgatgaca atgcaattat caccaccgac gttggtcagc atcagatgtg gaccgcgcaa 1200 gcttatccgc tcaatcgccc acgccagtgg ctgacctccg gtgggctggg cacgatgggt 1260 tttggcctgc ctgcggcgat tggcgctgcg ctggcgaacc cggatcgcaa agtgttgtgt 1320 ttctccggcg acggcagcct gatgatgaat attcaggaga tggcgaccgc cagtgaaaat 1380 cagctggatg tcaaaatcat tctgatgaac aacgaagcgc tggggctggt gcatcagcaa 1440 cagagtctgt tctacgagca aggcgttttt gccgccacct atccgggcaa aatcaacttt 1500 atgcagattg ccgccggatt cggcctcgaa acctgtgatt tgaataacga agccgatccg 1560 caggcttcat tgcaggaaat catcaatcgc cctggcccgg cgctgatcca tgtgcgcatt 1620 gatgccgaag aaaaagttta cccgatggtg ccgccaggtg cggcgaatac tgaaatggtg 1680 ggggaataa 1689

Claims (8)

A bacterial variant with increased isoleucine-producing ability, including inactivated acetohydroxyacid synthase I, wherein the strain replaces, inserts, deletes , or combines the nucleotide sequence of ilvB with acetohydride The strain characterized in that the hydroxy acid thinsaase I is inactivated strain.
delete According to claim 1, wherein the strain is a strain, characterized in that the strain reduced valine production capacity compared to wild-type bacterial strains.
According to claim 1, wherein the strain is a strain, characterized in that the ratio of the production of valine to the production of isoleucine 1-10%.
According to claim 1, wherein said strain is a strain of the genus Escherichia.
The method of claim 5, wherein the strain is Escherichia coli ), Escherichia albertii), Escherichia Blige state (Escherichia blattae), Escherichia peogu Sony (Escherichia fergusonii ) ( Escherichia hermannii ) or Escherichia vulgaris ( Escherichia) vulneris ) strain.
delete Isoleucine production comprising the step of inactivating acetohydroxy acid synthase I by substitution, insertion, deletion or combination thereof of the nucleotide sequence encoding ilvB of acetohydroxyacid synthase I Method for producing bacterial mutant strains having increased ability.

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