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CN-121975748-A - Alpha-ketoglutarate dehydrogenase mutant and application thereof

CN121975748ACN 121975748 ACN121975748 ACN 121975748ACN-121975748-A

Abstract

The invention belongs to the technical field of fermentation engineering, and particularly relates to an alpha-ketoglutarate dehydrogenase mutant and application thereof. The mutant or salt thereof has significantly reduced enzyme activity compared to the wild-type alpha-ketoglutarate dehydrogenase, so that the yield and/or productivity of citric acid can be improved when Aspergillus niger comprising the mutant or salt thereof is subjected to citric acid fermentation.

Inventors

  • YANG JINHUAN
  • WANG FANG
  • LI WEILI
  • Bi Duanduan
  • WEI JIE

Assignees

  • 安徽丰原发酵技术工程研究有限公司

Dates

Publication Date
20260505
Application Date
20260108
Priority Date
20250108

Claims (12)

  1. 1. An alpha-ketoglutarate dehydrogenase mutant having a mutation of V160P, T385H and/or I679R as compared with a wild-type alpha-ketoglutarate dehydrogenase, or a salt thereof.
  2. 2. A mutant according to claim 1, wherein, The amino acid sequence of the wild alpha-ketoglutarate dehydrogenase is shown as SEQ ID NO. 1; preferably, the amino acid sequence of the alpha-ketoglutarate dehydrogenase mutant is shown as SEQ ID NO. 5.
  3. 3. A nucleic acid molecule encoding the alpha-ketoglutarate dehydrogenase mutant according to any one of claims 1-2.
  4. 4. The nucleic acid molecule of claim 3, wherein, The nucleotide sequence of the nucleic acid molecule comprises: a1 Nucleotide sequence shown as SEQ ID NO. 6, or A2 Nucleotide sequence obtained by substituting and/or deleting and/or adding one or more nucleotides in SEQ ID NO. 6 and having the same function as the nucleic acid molecule shown in SEQ ID NO. 6, or A3 A nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 60%, 50%, 40% or 30% homology to SEQ ID NO.6 and having the same function as the nucleic acid molecule shown in SEQ ID NO. 6.
  5. 5. An expression cassette comprising the nucleic acid molecule of any one of claims 3-4.
  6. 6. A vector comprising the nucleic acid molecule of any one of claims 3-4 or the expression cassette of claim 5.
  7. 7. A cell comprising the alpha-ketoglutarate dehydrogenase mutant or salt thereof according to any one of claims 1-2, the nucleic acid molecule according to any one of claims 3-4, the expression cassette according to claim 5 or the vector according to claim 6, The cells are not involved in propagation material.
  8. 8. An aspergillus niger comprising the alpha-ketoglutarate dehydrogenase mutant or salt thereof according to any one of claims 1-2, the nucleic acid molecule according to any one of claims 3-4, the expression cassette according to claim 5, or the vector according to claim 6.
  9. 9. Use of the α -ketoglutarate dehydrogenase mutant according to any one of claims 1-2 or a salt thereof, the nucleic acid molecule according to any one of claims 3-4, the expression cassette according to claim 5, the vector according to claim 6, the cell according to claim 7 or the aspergillus niger according to claim 8 in any one of a 1) -a 2): a1 Preparing citric acid; a2 A product of synthesizing citric acid is prepared.
  10. 10. A method for preparing citric acid comprising the step of using the α -ketoglutarate dehydrogenase mutant or salt thereof according to any one of claims 1-2, the nucleic acid molecule according to any one of claims 3-4, the expression cassette according to claim 5, the vector according to claim 6, the cell according to claim 7 or the aspergillus niger according to claim 8.
  11. 11. The method of claim 10, wherein the step of determining the position of the first electrode is performed, Inoculating the Aspergillus niger according to claim 8 into a fermentation medium, and fermenting to obtain the Aspergillus niger; Preferably, the fermentation medium comprises a carbon source and a nitrogen source.
  12. 12. The method for constructing Aspergillus niger according to claim 8, wherein the nucleic acid molecule according to any one of claims 3 to 4 is introduced into wild-type Aspergillus niger.

Description

Alpha-ketoglutarate dehydrogenase mutant and application thereof Technical Field The invention belongs to the technical field of fermentation engineering, and particularly relates to an alpha-ketoglutarate dehydrogenase mutant and application thereof. Background Citric acid (CITRIC ACID) is also known as citric acid, and has the chemical name of 2-hydroxy propane tricarboxylic acid and 2-hydroxy propane-1,2,3-tricarboxylic acid (2-hydroxypropane-1, 2,3-tricarboxylic acid), and the molecular formula C 6H8O7. It is colorless transparent or semitransparent crystal, or granular, particulate powder, odorless, but has strong sour taste, but is pleasant, has little later astringency, is safe and nontoxic, and is widely used as a sour agent for foods and beverages. Fermentation processes are currently used industrially to produce citric acid. The breeding of high-yield citric acid strains plays an important role in the fermentation industry, and the research of the breeding of the citric acid production strain Aspergillus niger has been the focus of attention of researchers for decades. The metabolic pathway for producing citric acid by Aspergillus niger fermentation method is characterized by that the amylase produced during the growth and propagation of Aspergillus niger firstly converts the starch-containing raw material into glucose, then the glucose is converted into pyruvic acid by means of glycolysis (EMP) pathway, and the pyruvic acid can produce acetyl-CoA and CO 2 by means of pyruvic acid oxidase, then the acetyl-CoA and oxaloacetic acid can be acted on the citric acid synthetase (citric acid condensed enzyme) to synthesize the citric acid. The prior Aspergillus niger has high activity of alpha-ketoglutarate dehydrogenase, which results in reduced yield and productivity of citric acid. Disclosure of Invention The object of the first aspect of the present invention is to provide an alpha-ketoglutarate dehydrogenase mutant or a salt thereof. The object of the second aspect of the present invention is to provide a nucleic acid molecule encoding the α -ketoglutarate dehydrogenase mutant of the first aspect of the present invention. The object of a third aspect of the invention is to provide an expression cassette comprising a nucleic acid molecule of the second aspect of the invention. The object of the fourth aspect of the present invention is to provide a vector comprising the nucleic acid molecule of the second aspect of the present invention or the expression cassette of the third aspect. The object of the fifth aspect of the present invention is to provide a cell comprising the α -ketoglutarate dehydrogenase mutant of the first aspect of the present invention or a salt thereof, the nucleic acid molecule of the second aspect, the expression cassette of the third aspect or the vector of the fourth aspect. The sixth aspect of the present invention is directed to an aspergillus niger. The object of the seventh aspect of the present invention is to provide the use of the α -ketoglutarate dehydrogenase mutant of the first aspect of the present invention or a salt thereof, the nucleic acid molecule of the second aspect, the expression cassette of the third aspect, the vector of the fourth aspect, the cell of the fifth aspect or the A.niger. An object of an eighth aspect of the present invention is to provide a method for preparing citric acid. The ninth aspect of the present invention is directed to a method for constructing aspergillus niger according to the sixth aspect of the present invention. In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: in a first aspect of the invention, there is provided an alpha-ketoglutarate dehydrogenase mutant or a salt thereof, said alpha-ketoglutarate dehydrogenase mutant having a mutation of V160P, T385H and/or I679R as compared to the wild-type alpha-ketoglutarate dehydrogenase. In some embodiments, the amino acid sequence of the wild-type alpha-ketoglutarate dehydrogenase is shown as SEQ ID NO. 1. In some embodiments, the amino acid sequence of the alpha-ketoglutarate dehydrogenase mutant is shown as SEQ ID NO. 5. In a second aspect of the invention there is provided a nucleic acid molecule encoding an alpha-ketoglutarate dehydrogenase mutant of the first aspect of the invention. In some embodiments, the nucleotide sequence of the nucleic acid molecule comprises: a1 Nucleotide sequence shown as SEQ ID NO. 6, or A2 Nucleotide sequence obtained by substituting and/or deleting and/or adding one or more nucleotides in SEQ ID NO. 6 and having the same function as the nucleic acid molecule shown in SEQ ID NO. 6, or A3 A nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 60%, 50%, 40% or 30% homology to SEQ ID NO.6 and having the same function as the nucleic acid molecule shown in SEQ ID NO.6. In a third aspect of the invention there is provid