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Oleaginous yeast lipid droplet protein, coding gene and application thereof

A yeast lipid and protein technology, which is applied in the fields of application, genetic engineering, and plant gene improvement, can solve the problems of unpublished and unretrieved information on the sequence information of Rhodosporidium toruloides perilipidin, and improve the stability of lipid droplets, The effect of increasing the fat 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

As of the filing date of the patent, the amino acid sequence of this novel perilipin, the nucleotide sequence of its coding gene, and the results of subsequent functional research have not been published; NCBI has not retrieved any sequence about Rhodosporidium toruloides perilipin information

Method used

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  • Oleaginous yeast lipid droplet protein, coding gene and application thereof
  • Oleaginous yeast lipid droplet protein, coding gene and application thereof
  • Oleaginous yeast lipid droplet protein, coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1: Multi-omics study of Rhodosporidium toruloides

[0062] Whole-genome de novo sequencing of Rhodosporidium toruloides (R. toruloides): R. toruloides ATCC 10788 was cultured in YEPD medium at a temperature of 30°C and a rotational speed of 200 rpm for 48 hours. Bacteria were collected, and genomic DNA was prepared according to the standard genomic DNA extraction method (refined Molecular Biology Experiment Guide (Fourth Edition), Osper et al., translated by Yan Ziying, etc., published by Science Press), and analyzed by a solexa sequencer. Finally, 94 scaffolds (genome framework) were obtained, and the genome length was 20.2Mb.

[0063] R. toruloides transcriptome sequencing: R. toruloides ATCC10788 was cultured in a 2L fermenter at a temperature of 30°C and a pH of 5.6. The aeration rate was 100L / h, and the dissolved oxygen was maintained at 73% Linked with stirring), the YEPD medium flow acceleration rate is 0.828L / h (dilution rate is 0.53) (reactor working vol...

Embodiment 2

[0065] Example 2: Discovery of Rhodosporidium toruloides perilipin

[0066] R. toruloides CGMCC 2.1389 was cultured in liquid YEPD medium at 30℃ 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'.

[0067] R. toruloides CGMCC 2.1389 was fermented on nitrogen-limited medium to achieve oil accumulation. Carried out in a FUS-15L bioreactor with a liquid volume of 8L, an inoculum size of 10% (V / V), a temperature of 30°C, a ventilation rate of 0.8vvm, and dissolved oxygen controlled at 40-50% saturation value (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 ...

Embodiment 3

[0093] Embodiment 3: Cloning of Rhodosporidium toruloides rldp1 gene

[0094] Gene-specific primers were designed according to the nucleotide sequence of rldp1 as follows:

[0095] rldp1-Fw:5'-atggccaccgtcaacgagaagc-3'

[0096] rldp1-Rv:5'-tcactgcttctccccctcgcc-3'

[0097] The total RNA of R. toruloides CGMCC2.1389 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 =2.0, indicating good quality of total RNA. Total RNA samples were stored frozen at -80°C for later use.

[0098]First-strand cDNA was synthesized using High Fidelity PrimeScript RT-PCR Kit (purchased from Takara). 20 μl reaction system, first, 2 μl total RNA (~1 μg), 100 nM ...

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Abstract

The invention relates to an oleaginous yeast lipid droplet protein, coding gene and application thereof. The oleaginous yeast lipid droplet protein is the protein shown as the following (a) or (b): (a) a protein composed of an amino acid sequence shown by SEQ ID NO:1; and (b) a SEQ ID NO:1 derived protein that is obtained by subjecting the amino acid of SEQ ID NO:1 to substitution and / or deletion and / or adding by one or several amino acids and has related activity of the lipid droplet protein. Green fluorescent protein co-localization and grease fermentation analysis show that the oleaginous yeast lipid droplet protein and its coding gene provided by the invention can significantly improve the cellular grease accumulation and storage capacity. According to the invention, a recombinant engineering strain able to significantly improve grease accumulation can be obtained by heterologous expression of gene. The oleaginous yeast lipid droplet protein and its coding gene provided by the invention can be applied to construction of strains producing microbial oil and fatty acid derivatives, and can be used as the target for screening of drugs treating obese patients.

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 lipid droplet protein and the nucleotide encoding it are derived from Rhodosporidium toruloides. The invention also provides a method for constructing the recombinant cell for oil production. Background technique [0002] In the development of human civilization and science and technology, the production of fuels, bulk chemicals and fine chemicals mainly comes from traditional resources such as petroleum and coal. Although the depletion of energy resources can be supplemented by the development of new energy sources such as nuclear energy, wind energy, and solar energy, the production of chemicals must be based on the material basis of elements such as hydrocarbons or carbon, hydrogen, and oxygen. [0003] Grease is a renewable resource with low oxygen content, high ener...

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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