KR-20260066713-A - RECOMBINANT STRAIN FOR PRODUCING L-AMINO ACID, CONSTRUCTION METHOD THEREFOR, AND APPLICATION THEREOF

Abstract

The present invention provides a bacterium that produces L-amino acids, wherein the bacterium has improved expression of a polynucleotide encoding a protein represented by SEQ ID NO:3, improved expression of a polynucleotide encoding a protein represented by SEQ ID NO:31 and/or bases at the -45 bp and -47 bp sites of a promoter region represented by SEQ ID NO:57 are mutated. The invention further provides a polynucleotide encoding a protein, a recombinant vector comprising said polynucleotide sequence, a recombinant strain, and a method for producing L-amino acids, wherein the polynucleotide codes for a protein represented by SEQ ID NO:3 and arginine at the 334th site is substituted by a terminator, or codes for a protein represented by SEQ ID NO:31 and tyrosine at the 592nd site is substituted by phenylalanine, or bases at the -45 bp and -47 bp sites of a promoter region represented by SEQ ID NO:57 are mutated.

Inventors

• 웨이 아이잉
• 티엔 빈
• 가오 시아오항
• 멍 강
• 저우 시아오췬
• 자오 춘광
• 마 펑용
• 지아 후이핑
• 양 리펑
• 수 허우보
• 구어 시아오웨이

Assignees

• 이너 몽골리아, 에펜 바이오테크 컴퍼니 리미티드

Dates

Publication Date

20260512

Application Date

20201231

Priority Date

20200608

Claims (8)

1. In bacteria that produce L-lysine or L-glutamic acid, The expression of the polynucleotide encoding the amino acid sequence of SEQ ID NO:3 is improved; Here, a point mutation in the polynucleotide encoding the amino acid sequence of SEQ ID NO:3 is present, and the arginine at the 334th position of the amino acid sequence of SEQ ID NO:3 is substituted by a terminator, thereby enhancing expression; A bacterium that produces L-lysine or L-glutamic acid, characterized in that the above bacterium is Corynebacterium glutamicum .
2. In paragraph 1, Bacteria characterized by a polynucleotide encoding the amino acid sequence of SEQ ID NO:3 containing the nucleotide sequence of SEQ ID NO:1.
3. In paragraph 1, Bacteria characterized by the fact that the polynucleotide sequence having a point mutation while encoding the amino acid sequence of SEQ ID NO:3 includes the polynucleotide sequence represented by SEQ ID NO:2.
4. In polynucleotide sequences, It comprises a polynucleotide encoding an amino acid sequence indicated including SEQ ID NO:4; and/or A polynucleotide sequence characterized in that the above polynucleotide sequence includes a polynucleotide sequence represented by SEQ ID NO:2.
5. In terms of amino acid sequence, An amino acid sequence characterized by the above sequence being denoted by SEQ ID NO:4.
6. In recombination vectors, A recombinant vector characterized by including a polynucleotide sequence according to paragraph 4.
7. In the case of recombinant strains, A recombinant strain characterized by including a polynucleotide sequence according to paragraph 4.
8. In a method for producing L-lysine or L-glutamic acid, A method for producing L-lysine or L-glutamic acid, characterized by comprising the step of culturing a bacterium according to claim 1 or a recombinant strain according to claim 7 and recovering L-lysine or L-glutamic acid from said culture.

Description

Recombinant strain for producing L-amino acid, method of construction therefor, and application therefor The present application claims priority to the prior application filed with the National Intellectual Property Rights of China on October 15, 2020, with application number 202011105063.5 and titled "Recombinant strain producing L-amino acid and method of construction and application thereof"; the prior application filed with the National Intellectual Property Rights of China on August 7, 2020, with application number 202010790887.4 and titled "Recombinant strain producing L-lysine and method of construction and application thereof"; and the prior application filed with the National Intellectual Property Rights of China on June 8, 2020, with application number 202010514037.1 and titled "Recombinant strain with modified lysC gene and method of construction and application thereof". The three prior applications are cited in the present application in their entirety. The present invention belongs to the field of genetic engineering and microbial technology, and more specifically, relates to a recombinant strain that produces L-amino acids, a method for constructing the same, and its application. L-lysine has a wide range of applications in various fields, including medicine, food, and animal feed, with L-lysine used in feed additives accounting for more than 90% of the total. Currently, China is the second-largest consumer market for L-lysine and the largest producer of L-lysine. Currently, L-lysine is primarily produced by the direct fermentation method, which utilizes strains with a complete L-lysine biosynthetic pathway and produces it through aerobic fermentation using substrates such as waste molasses and starch hydrolysates. This method accounts for two-thirds of today's global L-lysine production and is a highly mature process that is widely distributed among microorganisms, primarily found in yeasts, bacteria, and fungi. Currently, the production strains used in the industry for L-lysine fermentation are mainly mutant strains bred from the genera Corynebacterium and Brevibacterium. With the development of metabolic and genetic engineering enabling the control of genetic mutations, it is now possible to improve L-lysine production by accurately identifying the key enzyme genes involved in L-lysine production during metabolic processes and increasing the expression of these major enzyme genes through the process of transforming starting strains using metabolic engineering. L-glutamic acid is primarily used in the production of monosodium glutamate, flavorings, salt substitutes, nutritional supplements, and biochemical reagents. L-glutamic acid itself can be used as a drug, participating in the metabolism of proteins and sugars in the brain and promoting oxidation processes. This product combines with ammonia in the body to form non-toxic glutamine, which reduces blood ammonia levels and alleviates symptoms of hepatic coma. In the past, the production of monosodium glutamate was mainly carried out through the hydrolysis of wheat gluten (gluten protein), but it is now utilized for large-scale production through microbial fermentation. The technical solution of the present invention is described in more detail below with reference to specific embodiments. It should be noted that the embodiments listed below are intended merely to illustrate and interpret the present invention and should not be interpreted as limiting the scope of protection of the present invention. All technologies embodied in the above description of the present invention fall within the scope of protection sought by the present invention. Unless otherwise specified, the raw materials and reagents used in the following embodiments are all commercially available products or can be manufactured by known methods, and the operations performed are all known in the art or can be carried out in accordance with the user guidelines of commercially available products. In the following examples, the basic medium used for the strains used in culture has the same composition, and necessary sucrose, kanamycin, or chloramphenicol, etc. corresponding to the components of the basic medium are added. The basic medium is composed as follows. In the following examples, the preparation and conditions of the SSCP electrophoresis PAGE are as follows. In the following examples, the fermentation medium and fermentation process of L-lysine are as shown in Tables 1 and 2. Table 1 L-Lysine Fermentation Medium Formulation Table 2. L-Lysine Fermentation Control Process In the following examples, the fermentation medium and fermentation process for L-glutamic acid are as shown in Tables 3 and 4. Table 3 L-Glutamic Acid Fermentation Medium Formulation Table 4. Fermentation Control Process of L-Glutamic Acid Example 1 Construction of a transform vector pk18- NCgl0609R334* containing a point-mutated NCgl0609 gene-coding region Based on the genome sequence of Corynebacte