KR-102960743-B1 - Microorganism of Corynebacterium genus producing L-glutamic acid and method for producing L-glutamic acid using the same

Abstract

The present invention relates to a microorganism of the genus Corynebacterium that produces L-glutamic acid and a method for producing L-glutamic acid using the same. Specifically, the invention relates to a variant of the transcription regulator IolR involved in the L-glutamic acid biosynthetic pathway, a polynucleotide, and a transformant, and a method for producing L-glutamic acid using the same. The transcription regulator IolR variant according to the present invention has altered protein activity by substituting one or more amino acids in the amino acid sequence constituting the transcription regulator IolR, so that a recombinant microorganism containing the same can efficiently produce L-glutamic acid.

Inventors

• 이자경
• 최원우
• 이상준
• 김석수

Assignees

• 대상 주식회사

Dates

Publication Date

20260507

Application Date

20230428

Claims (6)

1. delete
2. delete
3. A transformant having improved L-glutamic acid production ability compared to the wild type, comprising a transcription regulator IolR variant consisting of the amino acid sequence of SEQ ID NO. 2 or a polynucleotide encoding it.
4. In claim 3, The above transformant is a transformant that is a microorganism of the genus Corynebacterium .
5. In claim 3, The above polynucleotide is a transformant comprising the nucleotide sequence of SEQ ID NO. 1.
6. A method for producing L-glutamic acid comprising the step of culturing the transformant of claim 3 in a medium.

Description

Microorganism of the genus Corynebacterium producing L-glutamic acid and method for producing L-glutamic acid using the same The present invention relates to a microorganism of the genus Corynebacterium that produces L-glutamic acid and a method for producing L-glutamic acid using the same. Specifically, the invention relates to a variant of the transcription regulator IolR involved in the L-glutamic acid biosynthetic pathway, a polynucleotide, and a transformant, and a method for producing L-glutamic acid using the same. L-glutamic acid is a representative amino acid produced by microbial fermentation, and monosodium L-glutamate (MSG) is widely used as a seasoning for household use and processed food production because it balances and harmonizes the overall taste of food, thereby increasing the preference for foods such as meat, fish, chicken, vegetables, sauces, soups, and seasonings, and can enhance the taste of low-salt foods with up to 30% less salt. To briefly examine the fermentation pathway of L-glutamic acid, glucose primarily undergoes the glycolytic pathway, but some is metabolized into two molecules of pyruvate via the pentose phosphate pathway. One of these molecules fixes CO₂ to become oxaloacetic acid, while the other molecule combines with acetyl CoA from pyruvate to become citric acid. Oxaloacetic acid and citric acid then enter the citric acid cycle (TCA cycle) to become alpha-ketoglutaric acid. Here, the oxidative metabolic pathway for the oxidation of alpha-ketoglutarate to succinic acid is absent, and isocitrate dehydrogenase and glutamate dehydrogenase are closely involved, so the reductive amino acid reaction of alpha-ketoglutarate proceeds efficiently and L-glutamic acid is produced. L-glutamic acid production can be achieved using wild-type strains obtained from nature or mutant strains modified to enhance their glutamic acid production capabilities. Recently, to improve the efficiency of L-glutamic acid production, genetic recombination technology has been applied to microorganisms such as Escherichia coli and Corynebacterium, which are widely used for the production of useful substances like amino acids and nucleic acids. This has led to the development of various recombinant strains or mutants with superior L-glutamic acid production capabilities, as well as methods for producing L-glutamic acid using these strains. In particular, there have been attempts to increase L-glutamic acid production by targeting genes involved in the L-glutamic acid biosynthetic pathway, such as enzymes, transcription factors, and transport proteins, or by inducing mutations in promoters that regulate their expression. However, since the types of proteins—including enzymes, transcription factors, and transport proteins—directly or indirectly involved in L-glutamic acid production number in the tens to hundreds, much research is still needed regarding whether changes in the activity of these proteins lead to an increase in L-glutamic acid production capabilities. Figure 1 is the structure of a pK19msb plasmid according to one embodiment of 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. Preparation of a strain expressing a transcription regulator IolR variant To determine the effect of a variant (Sequence No. 2) in which the 146th leucine (L) in the amino acid sequence (Sequence No. 4) of transcription regulator IolR is substituted with phenylalanine (F) on the production of L-glutamic acid, a vector expressing the said transcription regulator IolR variant and a strain into which said vector was introduced were constructed. 1-1. Vector Construction for Expression of Transcription Regulator IolR Variant PCR was performed using the genomic DNA of wild-type Corynebacterium glutamicum ATCC13869 as a template, with primer pairs 1 and 2 and 3 and 4, respectively. Subsequently, overlapping PCR was performed using primer pairs 1 and 4 with the two PCR products as templates to ligate them into a single fragment. The PCR fragment and the pK19msb plasmid (SEQ No. 5) were treated with the restriction enzyme smaI (NEB) and cloned using T4 ligase. The constructed plasmid was named pK_iolR(L146F). For PCR, pfu premix (Bioneer) was used, and after denaturation at 95°C for 5 minutes, the reaction was repeated 30 times at 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute, followed by a reaction at 72°C for 5 minutes. The primer sequences used for plasmid construction are as shown in Table 1 below. Primer nameSequence numberPrimer sequence (5’-3’)Primer 16GAAGACCTCGACCGCAACGPrimer 27GAGCGGAAGCGGCGGATCPrimer 38GATCCGCCGCTTCCGCTCPrimer 49GGAGCGCTGCTGCATATC 1-2. Production of mutant strains introduced with the transcription regulator IolR variant Corynebacterium glutamicum U3 (KCCM13218P) was