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Alpha-ketoglutarate producing yeast engineering strain and construction method thereof

A technology of yeast engineering and construction method, which is applied in the field of genetic engineering, can solve problems affecting the distribution of flow direction, and achieve the effect of broad application prospects

Active Publication Date: 2013-05-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on how coenzyme A affects carbon metabolism and the flow direction distribution of carbon metabolism in industrial microorganisms

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1 Yarrowia lipolytica (Yarrowia lipolytica) WSH-Z06△ leu2 Strain construction and identification

[0040] According to the NCBI website Y. lipolytica of LEU2 Gene sequence (GenBank: M27209.1), design two pairs of primers (L1, L2 and R1, R2, see Table 1) to amplify LEU2 A fragment of about 1000bp upstream and downstream of the gene. In addition, using the pUG66 plasmid as a template, Ble-F and Ble-R as primers (see Table 1), PCR amplification with lox P sequence Ble The gene fragment is connected with pMD18-T simple vector after fusion PCR using the homologous sequence among the three fragments to obtain a recombinant plasmid. The recombinant plasmid was transformed into Escherichia coli JM109, and colonies that could grow on the Amp plate were picked for colony PCR identification with primers L1 and R2, and a band with a size of about 3000bp was obtained. Colony PCR verified that the correct transformants were extracted from the plasmid, and seq...

Embodiment 2

[0052] Construction and Identification of Example 2 Yeast Engineering Bacteria

[0053] Using mouse cDNA as a template, ACL-F and ACL-R (see Table 1) as primers, PCR amplified ACLs Target fragment (about 3500bp in size). Using In-Fusion PCR technology to ACLs The gene was connected with the plasmid pINA1269 digested with Pml I and BamH I to obtain ACLs Gene expression vector pINA1269-ACL, the constructed recombinant plasmid was digested and analyzed, and DNA sequencing was carried out. The results of gene sequencing were consistent with expectations, indicating that the recombinant plasmid was constructed correctly. The recombinant plasmid pINA1269-ACL was digested with Not I, purified, and transformed with lithium acetate Y. lipolytica WSH-Z06△ leu2 competent cells. The colonies that can grow on the MM plate were continuously transferred for three generations on the MM plate to obtain genetically stable yeast engineering progeny. Pick out several genomes of positive...

Embodiment 3

[0059] Embodiment 3 fermentation production

[0060] Inoculate the genetically engineered bacteria and the starting strains into the seed medium, add 50 ml of liquid in a 500 ml shaker flask, and culture at 28°C and 200 rpm for 20-24 h. The cultivated seeds were inserted into the fermentation medium according to the inoculation amount of 10%, and cultured at 28°C and 200 rpm for 144 h.

[0061] After 144 hours of fermentation, the comparison between the engineered yeast strain and the starting strain: (1) The a-KG yield in the starting strain was 35.2 g / L, and the a-KG yield in the engineered yeast strain could reach 45.3 g / L, which was 1.29 times that of the starting strain (2) The content of pyruvate in the starting bacteria was 25.0 g / L, while the content of pyruvate in the yeast engineering bacteria was 17.2 g / L, and the by-products were greatly reduced.

[0062] Seed medium (g / L): 20 g glucose, 10 g peptone, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate hep...

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Abstract

The invention discloses an alpha-ketoglutarate producing yeast engineering strain and a construction method thereof, which belong to the field of genetic engineering. In the method, a molecular approach is adopted, and the yeast engineering strain Y.lipolytica ACL with improved ATP-citrate lyase activity is obtained by over-expressing an ATP-ATP-citrate lyase ACL coding gene from a mouse into a strain Yarrowia lipolytica WSH-Z06 for producing alpha-ketoglutarate by a fermentation method. Compared with a parent strain, the genetic engineering strain provided by the invention can intracellularly accumulate acetyl coenzyme A with the dry cell weight (DCW) of 0.89 mM / g by adopting glycerol as the unique carbon source so as to achieve the ACL activity of 3.56 U / mg protein improved by 7.5 times, achieve the alpha-ketoglutarat yield of 45.3 g / L which is 1.29 times that of parent protein and reduce the pyruvic acid yield to 17.2 g / L which is 68.8 percent of that of the parent strain, and has vast application prospect.

Description

technical field [0001] The invention relates to an α-ketoglutarate yeast engineering bacterium and a construction method thereof, in particular to an engineering bacterium for producing α-ketoglutarate by overexpressing an ATP-citrate lyase gene, and belongs to the field of genetic engineering. Background technique [0002] a-ketoglutarate (a-KG), also known as a-gluconic acid, 2-oxoglutarate or a-carbonyl glutaric acid. Molecular formula is C 5 h 6 o 5 , The relative molecular weight is 146.1, a-KG is white or off-white crystal or crystalline powder, colorless and easily soluble in water. a-KG plays an important role in the metabolism of microbial cells and is one of the important intermediate products of the tricarboxylic acid cycle (TCA). It participates in the synthesis of amino acids, proteins, vitamins and energy metabolism in organisms. a-KG is widely used in industries such as food, medicine, organic synthesis, cosmetics and feed. [0003] The cofactors involved...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/60C12N15/79C12P7/50
Inventor 陈坚殷晓霞周景文堵国成
Owner JIANGNAN UNIV
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