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CN-121991866-A - Glutamic acid production strain and application thereof

CN121991866ACN 121991866 ACN121991866 ACN 121991866ACN-121991866-A

Abstract

The invention belongs to the technical field of biology, and particularly relates to corynebacterium glutamicum for producing L-glutamic acid, wherein BBD29_RS13520 protein in the strain cannot normally function. Compared with a wild strain, the strain with the BBD29_RS13520 protein incapable of functioning normally provided by the invention has the advantages that the growth of the strain under the high temperature condition is obviously enhanced, the yield and the conversion rate of the L-glutamic acid are both improved, the production cost of the L-glutamic acid can be reduced in production, and a new strategy is provided for large-scale production.

Inventors

  • ZHENG PING
  • SHI SHUZHI
  • LIU SHIZHOU
  • WANG FENG
  • SUN QINBO
  • Chen jiuzhou
  • DING HAIJIE
  • CAI NINGYUN
  • WANG JUNCHENG
  • SUN JIBIN
  • ZHOU WENJUAN
  • LIU YUANTAO
  • WANG YE

Assignees

  • 中国科学院天津工业生物技术研究所
  • 呼伦贝尔东北阜丰生物科技有限公司

Dates

Publication Date
20260508
Application Date
20241105

Claims (10)

  1. 1. Corynebacterium glutamicum producing L-glutamate, wherein the genetic manipulation is such that the protein of any one of the following corynebacterium glutamicum does not function normally: a) A protein with an amino acid sequence shown as SEQ ID NO. 1; B) A protein having an amino acid sequence homology of more than 90% with the protein of A) and having the same function as that of A); Specifically, the failure to function normally means that the protein is inactivated or expression is weakened to increase the L-glutamic acid yield and conversion rate of the strain.
  2. 2. Corynebacterium glutamicum producing L-glutamic acid according to claim 1, wherein the capacity to produce L-glutamic acid is increased by at least 10%, preferably by at least 20%, more preferably by at least 30%, more preferably by at least 40% relative to the starting strain.
  3. 3. Corynebacterium glutamicum producing L-glutamic acid according to claim 1, characterized in that the recombinant strain is obtained by knocking out or mutating the nucleic acid sequence encoding the bbd29_rs13520 protein by genetic manipulation, in particular by attenuating the gene expression or producing genes which are not expressed or which are not active or have reduced activity in spite of the expression, preferably the nucleic acid sequence encoding the bbd29_rs13520 protein is as shown in SEQ ID No. 2.
  4. 4. The corynebacterium glutamicum producing L-glutamate according to claim 1, wherein the starting strain is selected from corynebacterium glutamicum ATCC13869 and the L-glutamate producing strain obtained by engineering on the basis of this, which engineering comprises but is not limited to the enhancement or overexpression of one or more genes selected from the group consisting of: a. yggB gene encoding mechanically sensitive channel protein; b. Fxpk gene encoding phosphoketolase; c. The pyc gene encoding pyruvate carboxylase; d. A gdh gene encoding glutamate dehydrogenase; e. a gene encoding carbonic anhydrase; f. the gltA gene encoding malate synthase.
  5. 5. The corynebacterium glutamicum of claim 4, wherein said starting bacterium further comprises, but is not limited to, one or more genes selected from the group consisting of: a. an odhA gene encoding alpha-ketoglutarate dehydrogenase; b. amtR gene encoding a transcription regulatory gene; c. acnR gene encoding a transcriptional repressor.
  6. 6. The corynebacterium glutamicum according to claim 5, wherein said strain is further modified, wherein the NCgl1221 homologous gene is introduced with the A111V mutation, the GltA encoding gene is introduced with the C361Y mutation, the gltA gene promoter is introduced with the P gltA-Ts24 mutation, and the gdh gene promoter is introduced with the P gdh-RBS19 mutation, and preferably said strain is corynebacterium glutamicum SCgGC.
  7. 7. Use of the corynebacterium glutamicum producing L-glutamic acid according to any one of claims 1 to 6 for the preparation of L-glutamic acid.
  8. 8. A method for producing L-glutamic acid using the corynebacterium glutamicum producing L-glutamic acid according to any one of claims 1 to 6, wherein the corynebacterium glutamicum producing L-glutamic acid is cultured by fermentation according to any one of claims 1 to 6.
  9. 9. The method of claim 8, wherein the fermentation medium is glucose, 80 g/L, KH 2 PO 4 , 1 g/L, urea, 10 g/L, or corn steep liquor dry powder ,5 g/L;MgSO 4 ·7H 2 O,0.4 g/L;FeSO 4 ·7H 2 O,10 mg/L;MnSO 4 ·4H 2 O,10 mg/L;VB 1 ,200 μg/L;MOPS,40 g/L;, initial pH7.5.
  10. 10. The method of claim 8, further comprising the step of further isolating and purifying the produced L-glutamic acid.

Description

Glutamic acid production strain and application thereof Technical Field The present invention relates to the field of biotechnology. In particular, the invention relates to an L-glutamic acid high-producing strain constructed by inactivation or expression attenuation of BBD29_RS13520 protein and application thereof. Background L-glutamic acid is the traditional amino acid variety with the largest yield at present, is a basic substance for forming protein required by animal nutrition, plays an important role in the protein metabolism process in organisms, is widely applied to the fields of medicine, chemical industry, livestock and the like, and is a biological fermentation product with the largest production standard in China due to high demand and high yield. Currently, L-glutamic acid is produced mainly by microbial fermentation, and commonly used industrial fermentation microorganisms include strains of Corynebacterium (Corynebacterium) and Brevibacterium (Brevibacterium). Corynebacterium glutamicum (Corynebacterium glutamicum) has become the most important glutamic acid-producing strain in industry due to its physiological superiority. Along with the development of synthetic biology technology, reports of improving the glutamic acid yield of corynebacterium glutamicum by metabolic engineering are gradually increased, such as strengthening the glutamic acid yield of a strain with the expression of a key enzyme for synthesizing L-glutamic acid, namely, improving the glutamic acid yield of a strain (WO 0053726A 1), strengthening the four-carbon back-fill pathway to maintain the efficient synthesis of L-glutamic acid, strengthening the expression of an L-glutamic acid efflux protein MscCG and improving the yield of L-glutamic acid, and the like (Wang Y,Cao G,Xu D,et al.A novel Corynebacterium glutamicum L-glutamate exporter.Applied and Environment Microbiology,2018,84(6):e02691–02617)., although the means can improve the L-glutamic acid yield of the strain, due to the fact that the L-glutamic acid synthesis pathway is shorter, target transformation points and strategies can be limited relatively, the difficulty of further improving the level of industrial strains is great, and the field still needs to deeply develop target research related to the synthesis of L-glutamic acid so as to further improve the fermentation level of the industrial strain and improve the productivity of L-glutamic acid. The prior art shows that the membrane wall structure of corynebacterium glutamicum has a great influence on the synthesis and secretion of glutamic acid. In bacteria and fungi, however, there is a GH76, LAM hydrolase (Lipoarabinomannan Hydrolase, lamH), the primary activity of which is alpha-mannanase, which can degrade alpha-1, 6-mannosidic linkages, such as those in the alpha-1, 6-linked backbones of fungal mannoprotein and mycobacterial cell wall lipomannans (lipomannan, LM), lipoarabinomannan (lipoarabinomannan, LAM) and phosphatidylinositol mannans. Recently Frankli et al identified a LAM hydrolase in mycobacteria that specifically cleaves the alpha-1, 6-mannoside linkage within LM and LAM, producing acetylated phosphatidylinositol mannose (ACETYLATED PHOSPHATIDYLINOSITOL MANNOSIDES, acPIM 2) and arabinomannan (arabinomannan, AM). Knocking-out LamH can lead to LM and LAM accumulation, the AM and AcPIM2 content is reduced, and simultaneously, the expression of LAM synthesis pathway gene is regulated downwards, further research shows that LamH deletion can prolong the delay period of the strain, and the product AM supplemented with LamH can effectively trigger the strain to exit the delay period and enter the logarithmic growth phase, so that AM can be used as a molecular signal for mycobacterium growth, weakening LamH can influence the mycobacterium growth (The mycobacterial glycoside hydrolase LamH enables capsular arabinomannan release and stimulates growth.Nat Commun.2024,15:5740.). in macrophages, however, at present, whether the protein exists in corynebacterium glutamicum or not, and whether the protein can promote the L-glutamic acid yield and the conversion rate of the strain is unknown. Disclosure of Invention The cell membrane wall structure of Corynebacterium glutamicum is similar to that of Mycobacteria and contains a series of non-covalently bound PIM (phosphatidyl-myo-inositol mannosides) type glycolipids, LM and LAM type lipopolysaccharides. Through genomic sequencing and annotation analysis, we speculate that in corynebacterium glutamicum, bbd29_rs13520 (also known as bbd29_13515 protein) may also specifically cleave α -1, 6-mannosidic bonds within LM and LAM to produce AcPIM and AM, regulate the growth of corynebacterium glutamicum and affect cell membrane wall structure. Given the large impact of the membrane wall structure of corynebacterium glutamicum on glutamate synthesis and secretion, it is speculated that bbd29_rs13520 attenuation or knockout favors glutamate production. Further, experime