Search

CN-122011138-A - ArcA mutant and application thereof

CN122011138ACN 122011138 ACN122011138 ACN 122011138ACN-122011138-A

Abstract

The invention belongs to the technical field of strain genetic engineering. The invention provides an arcA mutant, which is mutated into a stop codon at 94 th glutamic acid of arcA protein. The invention provides an engineering strain, which reduces acetic acid generation by about 90% in fermentation reaction, and shortens multiplication time by 10%.

Inventors

  • ZHANG YANPING
  • ZHAO LEI
  • LI YIN

Assignees

  • 中国科学院微生物研究所

Dates

Publication Date
20260512
Application Date
20241110

Claims (5)

  1. An ArcA mutant characterized by a mutation to the stop codon at the 94 th glutamic acid of ArcA protein.
  2. 2. An engineered strain comprising the ArcA mutant of engineering strain BW25113 * .
  3. 3. The method for constructing an engineering strain according to claim 2, wherein the recombinant strain is obtained by synthesizing an arcA upstream 500 bp-mutated arcA (E94 x) -sequence-arcA downstream 500bp, and transferring up-arcA (E94 x) -down as a template to a strain BW25113-Cm R -pCas9 together with pTraget-Cm R -N20; The strain BW25113-Cm R -pCas is obtained after up-Cm R -down and pTraget-arcA-N20 are transformed into the strain BW25113pCas, the strain BW 25113-pCas 9 is obtained after pCas9 plasmid is transformed into the strain BW25113, the up-Cm R -down is obtained after the upstream and downstream fragments of arcA genes are connected through a resistance gene Cm R , and the pTraget-arcA-N20 is obtained after the arcA N20 is designed by using online gRNA-N20; The pTraget-Cm R -N20 is obtained after N20 of Cm R resistance gene is designed by using online gRNA-N20; the arcA (E94) sequence is obtained after mutation of the 94 th glutamic acid of arcA protein to a stop codon.
  4. 4. Use of an ArcA mutant according to claim 1 for reducing acetate production.
  5. 5. Use of the engineered strain according to claim 2 for reducing acetic acid production.

Description

ArcA mutant and application thereof Technical Field The invention belongs to the technical field of strain genetic engineering. Background The TCA cycle (also known as the citric acid cycle or the tricarboxylic acid cycle) has important physiological and metabolic functions in microorganisms, which are key links in cellular metabolism, connecting multiple metabolic pathways such as glycolysis, oxidative phosphorylation, and amino acid and lipid synthesis. The TCA cycle is the central pathway for energy by aerobic respiration. NADH and FADH 2 are formed by oxidation of acetyl-CoA, and these molecules subsequently enter the electron transfer chain, driving oxidative phosphorylation to ATP. Intermediates of the TCA cycle (e.g., citric acid, α -ketoglutaric acid, oxaloacetic acid) are used not only for energy metabolism but also as carbon backbones for synthesis of amino acids, biomolecules and lipids. The TCA cycle links the glycolytic pathway with the electron transport chain, helping to regulate the energy requirements of the strain in different environments, especially the transition between aerobic and anaerobic conditions. Based on the significance, the TCA cycle is regulated and controlled at multiple levels, so that the strain is ensured to be metabolized and survive efficiently under different environmental conditions. ArcA (Aerobic Respiratory Control Regulator A) is a key regulator in E.coli that regulates aerobic/anaerobic respiration. ArcA and its chaperones ArcB constitute a two-component regulatory system that responds to changes in oxygen levels, more broadly, arcAB has been demonstrated to respond to changes in intracellular redox levels. ArcB activates ArcA by autophosphorylation in a hypoxic or intracellular state, and ArcA is subsequently phosphorylated and acts as a transcription repressor on a variety of genes involved in aerobic respiration. ArcA consists of two main functional domains and is activated by phosphorylation of its chaperone ArcB. The N-terminal response Regulatory Domain (RD) is located at the N-terminal of ArcA, and the C-terminal DNA Binding Domain (DBD) comprises a DNA binding domain. When ArcA is phosphorylated, the DNA binding domain at the C-terminus is able to bind to the promoter region of the target gene, regulating the expression of genes associated with aerobic respiration. ArcA phosphorylates to down regulate transcription of the key enzyme gene of the TCA cycle, and reduces TCA cycle activity, thereby turning to fermentation metabolism. Under aerobic or redox balance conditions, arcA is in a non-phosphorylated state, expression of TCA cycle-related genes is increased, and TCA cycle is activated to maximize energy production. Under some stress conditions, the overall transcriptional level of the TCA cycle is down-regulated due to limitations such as carbon starvation and redox imbalance, failing to provide adequate energy and substance precursor supply. Disclosure of Invention In view of this, the present invention provides an ArcA mutant. The mutant can be used for obviously improving the TCA cycle efficiency under certain stress conditions, such as formic acid is the only carbon source. The ArcA mutant provided by the invention is obtained by mutating 94 th glutamic acid of ArcA protein into a stop codon. The direct effect of ArcA on global regulation by removal of the DNA binding domain is greatly diminished, but can still be involved in the perception and signaling of redox status and may affect the redox balance of the cell by protein-protein interactions. These non-DNA binding functions allow ArcA to continue to function to some extent as a redox sensor even though it is no longer directly regulating gene expression. Further, an engineering strain is provided, and engineering strain BW25113 containing the ArcA mutant is provided. The invention also provides a construction method of the engineering strain, which is obtained by synthesizing arcA upstream 500 bp-mutated arcA (E94 x) sequence-arcA downstream 500bp, and transferring up-arcA (E94 x) -down as a template together with pTraget-Cm R -N20 into a strain BW25113-Cm R -pCas 9; the strain BW25113-Cm R -pCas9 is obtained after up-Cm R -down and pTraget-arcA-N20 are transformed into the strain BW25113pCas9, the strain BW25113pCas is obtained after pCas plasmid is transformed into the strain BW25113, up-Cm R -down is obtained after upstream and downstream fragments of arcA gene are connected through a resistance gene Cm R, pTraget-arcA-N20 is obtained after an arcA N20 is designed by an online gRNA-N20, pTraget-Cm R -N20 is obtained after an arcA resistance gene N20 is designed by an online gRNA-N20, and the arcA (E94) sequence is obtained after an arcA protein 94 th glutamic acid is mutated to a stop codon. Further, the application of the ArcA mutant in improving the biological conversion efficiency of formic acid is also provided. Further, the application of the engineering strain in reducing acetic acid p