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R.glutinis lipid droplet protein 1, coding gene and application thereof

A yeast lipid and protein technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problem of increasing the oil content of recombinant strains, and achieve the effect of improving the stability of lipid droplets and increasing the oil content

Active Publication Date: 2014-08-06
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The researchers found that genes encoding isocitrate dehydrogenase (IDH) and malic enzyme (ME) from oleaginous microorganisms (Rhodosporidium toruloides and Liposporidium steerii) were expressed during oil accumulation in indigenous bacteria cerevisiae functional complementation analysis also proved that these genes can increase the oil content of recombinant strains, but failed to successfully transform a non-oleaginous strain into an oleaginous strain [Yang F, Zhang SF, Zhou YJ, Zhu ZW, Lin XP, Zhao ZK.Appl.Microbiol.Biotechnol.2012, 94(4), 1095-1105]

Method used

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  • R.glutinis lipid droplet protein 1, coding gene and application thereof
  • R.glutinis lipid droplet protein 1, coding gene and application thereof
  • R.glutinis lipid droplet protein 1, coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1: Discovery of Rhodotorula viscosus perilipin-like protein

[0062] R.glutinis ATCC204091 was cultured in liquid YEPD medium at 30°C for 36h. The wet cells were collected by centrifugation, washed twice with distilled water, and stored in a -80°C refrigerator for RNA and protein extraction. The same cultured samples (parallel samples) were used to determine the dry cell weight and oil content. Sample named 'YEPD'.

[0063] R. glutinis ATCC204091 was fermented on nitrogen-limited medium to achieve oil accumulation. It is carried out in a FUS-15L bioreactor with a liquid volume of 8L, an inoculum volume of 10%, a temperature of 30°C, an aeration volume of 0.8vvm, and dissolved oxygen controlled at 40-50% saturation (dissolved oxygen and stirring linkage). The pH was automatically controlled at 5.6 by dropwise addition of 10.0M NaOH and 2M HCl. Take out 50mL of cell culture fluid cultured for 24h and 96h respectively, and the samples are named '24h' and '96h' ...

Embodiment 2

[0086] Example 2: Cloning of Rhodotorula viscosus rgldp1 gene

[0087] According to the amino acid sequence of RgLDP1, degenerate primers were designed:

[0088] rgldp1-sence: 5'-ATGGCNACNGTNAAYGA-3' (N: A / C / G / T, Y: C / T, / stands for "or")

[0089] rgldp1-anti: 5'-YYAYTCYTTYTCAGTGGT-3' (N: A / C / G / T, Y: C / T) ( / stands for "or")

[0090] The primers were all in the 5' to 3' direction.

[0091] The total RNA of R. glutinis ATCC204091 was extracted by liquid nitrogen grinding plus RNAiso method [Yang F, Tan HD, Zhou YJ, Lin XP, Zhang SF. Mol. Biotechnol. 2010, 47(2), 144-151]. The RNA was subjected to 1.5% agarose gel electrophoresis, observed and identified using a fluorescence-ultraviolet analyzer, and two clear bands could be seen. Analyze the total RNA sample with a UV / Vis spectrometer and measure the OD 260 / 280 =1.9, indicating good quality of total RNA. Total RNA samples were stored frozen at -80°C for later use.

[0092] First-strand cDNA was synthesized using High Fid...

Embodiment 3

[0094] Example 3: Prokaryotic expression and protein purification of Rhodotorula viscosus rgldp1 gene

[0095] According to the nucleotide sequence of rgldp1, the primers with corresponding restriction sites were designed (the underlined part of the primer rgldp1-EcoRI-F is the EcoRI restriction site, and the underlined part of the primer rgldp1-NotI-R is the NotI restriction site), The sequence is as follows:

[0096] rgldp1-EcoRI-F:G GAATT CAACATGGCCACCGTCAACGAGAAGC

[0097] rgldp1-NotI-R: AAAAGGAAAA GCGGCCGC TTACTCCTTTCTCAGTGGTCTCGAC

[0098] The primers were all in the 5' to 3' direction.

[0099]Using the cloning vector pMD18T-rgldp1 constructed in Example 2 as a template, use the primers rgldp1-EcoRI-F and rgldp1-NotI-R to amplify the sequence of the rgldp1 coding region. The pET28a vector (recycling large fragments for ligation reaction) was transformed into E.coli DH5a chemically competent cells, and the correctly constructed recombinant plasmid was named pET28a...

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Abstract

The present invention relates to R.glutinis lipid droplet protein 1, a coding gene and an application thereof. The R.glutinis lipid droplet protein 1 is the following protein (a) or (b), wherein the protein (a) is a protein comprising the amino acid sequence represented by SEQ ID NO:1, and the protein (b) is a protein obtained by carrying out substitution and / or deletion and / or addition of one or a plurality of amino acids on the amino acid represented by SEQ ID NO:1, has the droplet protein related activity, and is derived from the SEQ ID NO:1. Results of green fluorescent protein co-localization and grease fermentation analysis demonstrate that the cell grease accumulation and storage amount can be significantly increased with the R.glutinis lipid droplet protein 1 and the coding gene thereof. According to the present invention, a recombinant engineering strain capable of significantly increasing the grease accumulation amount is obtained through heterologous expression of the gene. The R.glutinis lipid droplet protein 1 and the coding gene can be applied for construction of microorganism grease and fatty acid derivative production strains, and can further be used as a target for screening obesity patient treatment drugs.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to an oleaginous yeast lipid droplet protein and its gene and application. Specifically, the oleaginous yeast lipoprotein and the nucleotide encoding it are derived from Rhodotorula glutinis (R. glutinis). The invention also provides a method for constructing the recombinant cell for oil production. Background technique [0002] Grease is a renewable resource with low oxygen content, high energy density, and moderate carbon chain length. It can replace fossil resources as the basic processing raw material for the chemical industry and renewable energy industry. It is a transition from a carbon-hydrogen economy to a carbon-hydrogen economy. The important link of its market potential is huge. Some microorganisms in nature can store more than 20% of the dry cell weight of oil in the cell under certain conditions (such as nitrogen source deficiency), among which triglyceride is the ...

Claims

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Application Information

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IPC IPC(8): C07K14/39C12N15/31C12N15/63C12N1/13C12N1/19C12N1/21
CPCC07K14/39
Inventor 赵宗保朱志伟张素芳
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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