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KR-102960744-B1 - Novel variant of PTS system EI component and transformant comprising the same

KR102960744B1KR 102960744 B1KR102960744 B1KR 102960744B1KR-102960744-B1

Abstract

The present invention relates to a novel variant of a PTS system EI component and a transformant containing the same, wherein the protein activity of the PTS system EI component variant is altered by substituting one or more amino acids among the amino acid sequences constituting the PTS system EI component, thereby allowing for the acquisition of a recombinant microorganism having a fructose-preferring characteristic.

Inventors

  • 최민진
  • 이한진
  • 홍인표
  • 이지하
  • 김하은
  • 박은서
  • 이선희

Assignees

  • 대상 주식회사

Dates

Publication Date
20260507
Application Date
20240404

Claims (6)

  1. A variant of the PTS system EI component composed of the amino acid sequence of SEQ ID NO. 4, wherein the 108th glutamic acid in the amino acid sequence of SEQ ID NO. 2 is substituted with lysine, the 137th arginine is substituted with cysteine, and the 166th alanine is substituted with threonine.
  2. A polynucleotide encoding a variant of claim 1.
  3. A transformant comprising a variant of claim 1 or a polynucleotide of claim 2.
  4. In claim 3, The above transformant is a transformant that is a microorganism of the genus Corynebacterium .
  5. In claim 3, The above-mentioned transformant is a transformant having a fructose-specific preference characteristic.
  6. A culture method comprising the step of culturing the transformant of claim 3 in a medium containing fructose.

Description

Novel variant of PTS system EI component and transformant comprising the same The present invention relates to a novel variant of a PTS system EI component and a transformant containing the same. The genus Corynebacterium is a microorganism that, along with Escherichia coli , has recently been primarily used to produce useful substances such as amino acids and nucleic acids. In particular, Corynebacterium glutamicum is widely used industrially because it possesses characteristics that facilitate genetic engineering and mass cultivation for commercial use, as well as high stability (Korean Registered Patent No. 10-0838038, Korean Registered Patent No. 10-2139806). Corynebacterium glutamicum can grow by utilizing glucose, fructose, sucrose, maltose, etc., as carbon and energy sources. As a characteristic of its carbon metabolism, when cultured on a substrate containing a mixture of various carbon sources, it lacks glycogen specificity and produces target products by non-selectively using a given carbon source, such as glucose or fructose. In other words, Corynebacterium glutamicum possesses the characteristic of simultaneously metabolizing fructose and glutose ( Kor. J. Microbiol. Biotechnol . Vol. 38, No. 4, 349-361 (2010)). The inventors induced chemical mutations in wild-type Corynebacterium glutamicum to obtain a mutant microorganism or mutant strain having a preference characteristic for a specific glycogen, and as a result, obtained a mutant strain favorable for fermentation using fructose, thereby completing the present invention. The present invention will be described in more detail below. However, this description is provided merely as an example to aid in understanding the invention, and the scope of the invention is not limited by this exemplary description. Example 1. Induction of NTG mutation To produce a mutant strain with a preference for a specific glycogen, wild-type Corynebacterium glutamicum was treated with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) to induce mutations. Corynebacterium glutamicum ATCC13032 was cultured in LB medium for 16 hours (30℃, 200 rpm). After culture, the cells were centrifuged at 4500 rpm for 10 minutes and suspended in saline/TM buffer. After resuspending the cells in saline/TM buffer, 100 µg/ml of NTG was added to induce mutation at 30℃, 200 rpm for 30 minutes. After repeating the above mutation induction process, the cells were suspended in 3 ml of distilled water and plated in CGXII minimal medium containing 2% fructose and 1% glucose (20 g/L fructose, 10 g/L glucose, 20 g/L ( NH₄ ) ₂SO₄ , 5 g /L urea, 1 g/L KH₂PO₄ , 1 g/ L LK₂HPO₄ , 0.25 g/L MgSO₄·7H₂O , 10 mg/L CaCl₂, 10 mg /L FeSO₄· 7H₂O , 10 mg /L MnSO₄ · H₂O , 1 mg/L ZnSO₄ · 7H₂O , 0.2 mg/L CuSO₄ · 5H₂O, 0.02 mg/L NiCl₂ 2 · 6H₂O , 0.2 mg/L biotin, 30 mg/L 3,4-dihydroxybenzoic acid, 21 g/L 3-morpholinopropanesulfonic acid (MOPS), and agar (pH 7.0) were plated and incubated at 30°C for 2 days for primary incubation. Strains forming a single colony were isolated and sterilized in CGXII minimal medium containing 2% fructose (20 g/L fructose, 20 g/L ( NH₄ ) ₂SO₄ , 5 g/L urea, 1 g/L KH₂PO₄ , 1 g/ L LK₂HPO₄ , 0.25 g/L MgSO₄ · 7H₂O , 10 mg/L CaCl₂, 10 mg/L FeSO₄·7H₂O , 10 mg/ L MnSO₄· H₂O , 1 mg/L ZnSO₄ · 7H₂O , 0.2 mg/L CuSO₄ · 5H₂O , 0.02 mg/L NiCl₂ · 6H₂O , 0.2 mg/L biotin, 30 mg/L 3,4-dihydroxybenzoic acid and 21 Fructose-preferring mutant strains were selected by secondary culture in g/L 3-morpholinopropanesulfonic acid (MOPS); pH 7.0). WGS analysis was performed on the selected mutant strains. As a result, it was confirmed that three amino acid variations (E108K, R137C, and A166T) occurred in the amino acid sequences of the EI components of the PTS system. Subsequently, three variants of the PTS system EI component (E108K, R137C, and A166T) were used to produce mutant strains with fructose preference characteristics. Example 2. Preparation of a strain expressing a PTS system EI component variant To determine the effect on fructose availability of a variant (Sequence No. 4) in which glutamic acid at the 108th position and alanine at the 166th position are substituted with lysine, arginine at the 137th position is substituted with cysteine, and alanine at the 166th position is substituted with threonine in the amino acid sequence of the PTS system EI component (Sequence No. 2), a vector expressing the PTS system EI component variant and a strain into which the vector was introduced were constructed. 2-1. Production of Transgenic Vectors Using the genomic DNA of the Corynebacterium glutamicum ATCC13032 mutant strain, which was conferred fructose preference due to NTG treatment in Example 1, as a template, regions with three amino acid mutations (E108K, R137C, and A166T) in the amino acid sequence of the PTS system EI component were amplified by PCR using primers 1 and 2. All PCRs were performed using pfu premix (bioneer), and after denaturation at 95°C for 5 minutes, the reaction was repeated 30 times for 30 s