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Citric acid transport protein and application thereof in lipid synthesis

A transporter, citric acid technology, applied in the field of genetic engineering and microbial engineering

Active Publication Date: 2021-04-16
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the increasing demand for microbial oils, and the oil production capacity of existing oil-producing microorganisms cannot meet this demand, how to further increase the oil production capacity of microorganisms has become an urgent problem to be solved

Method used

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  • Citric acid transport protein and application thereof in lipid synthesis
  • Citric acid transport protein and application thereof in lipid synthesis
  • Citric acid transport protein and application thereof in lipid synthesis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Screening of genes encoding citrate-ketoglutarate transporters

[0041] Specific steps are as follows:

[0042] Using the YALI0 lipolytic citrate-ketoglutarate transporter gene sequence identified in NCBI (NCBI ID: YALI0B10736p) as a template, BLAST comparison was performed in the gene bank of the sequenced M.alpina ATCC 32222 strain According to the condition of homology>30%, an alternative target gene sequence (MA-00120-22) was obtained; the final target gene was named MaYHM (nucleotide sequence as shown in SEQ ID No.2), The corresponding protein is named MaYHM (the amino acid sequence is shown in SEQ ID No.1).

[0043] The full-length corresponding cDNA of MaYHM is 930bp, encoding 309 amino acids. In order to further judge whether the screened MaYHM belongs to the citrate-ketoglutarate transporter, amino acid homology and conservative structure analysis were carried out between it and the citrate-ketoglutarate transporter identified in NCBI.

[0044] Su...

Embodiment 2

[0045] Example 2: Cloning of MaYHM

[0046] Specific steps are as follows:

[0047] The total RNA of Mortierella alpina (Mortierella alpina) ATCC 32222 was extracted using the TRIzol method, and cDNA was obtained by reverse transcription according to the instructions of the Thermo reverse transcription kit, and amplified by PCR reaction in the cDNA library of Mortierella alpina (Mortierella alpina) ATCC 32222. Add MaYHM, and the primers used to amplify MaYHM are listed in Table 1.

[0048] The PCR instrument used is BIO-RAD T100 Thermal Cycler, using KOD plus high-fidelity DNA polymerase, the reaction system is 50 μL, and the content of the system is carried out according to the instructions of the DNA polymerase; the reaction process is as follows: pre-denaturation at 95°C for 5 minutes, then denaturation at 95°C 30s, annealing at 55°C for 30s, extension at 68°C for 1.5min, repeat the above three steps 30 times, then fully extend at 68°C for 5min, and finally drop to 12°C fo...

Embodiment 3

[0052] Embodiment 3: Construction of overexpressing MaYHM recombinant bacteria

[0053] Specific steps are as follows:

[0054] (1) Construction of Mortierella alpina expression vector

[0055] The MaYHMDNA fragment obtained in Example 2 and the expression vector pBIG2-ura5s-ITs were digested using restriction endonucleases XhoI and KpnI, and then utilized T 4 Ligase ligated the digested and purified MaYHM DNA fragment with pBIG2-ura5s-ITs to obtain a ligation product. The specific enzyme digestion system (20 μL) is shown in Table 2.

[0056] Table 2 enzyme digestion system

[0057] Reagent Dosage 10×cutmart buffer 2μL restriction endonuclease 1μL PCR product or vector 200ng~1μg wxya 2 o

Make up to 20μL

[0058] After connecting the obtained ligation product overnight at 4-16°C, transform it into Escherichia coli TOP10 competent cells. The transformation method is as follows: take 100 μL of competent cells in a sterile state, ...

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Abstract

The invention discloses a citric acid transport protein and application thereof in lipid synthesis, belonging to the technical field of genetic engineering and microbial engineering. The citric acid-ketoglutarate transport protein with an amino acid sequence shown as SEQ ID No. 1 has the function of promoting microorganisms to produce fatty acid; after recombinant mortierella alpine containing the citric acid-ketoglutarate transport protein is subjected to shaking culture for 7 days, the fatty acid content of mortierella alpine containing the citric acid-ketoglutarate transport protein can reach 36.1% of the dry weight of cells, and is increased by 30.4% compared with the fatty acid content of mortierella alpine not containing the citric acid-ketoglutarate transport protein. Through a genetic engineering means, the lipid synthesis accumulation capacity of oil-producing microorganisms such as mortierella alpine is improved on a gene level, and meanwhile, the normal growth of the oil-producing microorganisms can be ensured, so a technical support is provided for more efficient microbial oil industrial production.

Description

technical field [0001] The invention relates to a citrate transporter and its application in lipid synthesis, belonging to the technical fields of genetic engineering and microbial engineering. Background technique [0002] With the continuous popularization of the concept of healthy diet, people's demand for polyunsaturated fatty acids continues to increase. However, with the reduction of arable land and the pollution of the marine environment, plant, animal and algae oils have been unable to meet people's needs. Microbial oil is another edible oil developed by humans after vegetable oil and animal oil. Some oleaginous microorganisms can accumulate abundant polyunsaturated fatty acids (PUFAs), such as EPA, AA, etc. Eating an appropriate amount of microbial oils rich in PUFAs can significantly reduce the lipid and cholesterol levels in the plasma of patients with hyperlipidemia and hypercholesterolemia, while increasing the levels of apolipoprotein and high-density lipopro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/37C12N15/80C12N15/70C12N15/74C12N15/31C12N1/21C12P7/64C12R1/645C12R1/19C12R1/01
Inventor 唐鑫陈海琴凌凤珠赵建新张灏陈永泉陈卫
Owner JIANGNAN UNIV
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