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Rhodotorula glutinis oil genetic engineering strain and construction method and application thereof

A technology of genetically engineered bacteria, Rhodotorula viscosus, applied in the field of construction of Rhodotorula viscosus high-yielding genetically engineered bacteria

Active Publication Date: 2012-11-28
广州溯原生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This is because although there are many NADPH-generating processes in microbial metabolic pathways, FAS can almost only use NADPH produced by ME (Wynn JP, et al. The role of malic enzyme in the regulation of lipid accumulation in filamentous fungi. Microbiology, 1999)

Method used

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  • Rhodotorula glutinis oil genetic engineering strain and construction method and application thereof
  • Rhodotorula glutinis oil genetic engineering strain and construction method and application thereof
  • Rhodotorula glutinis oil genetic engineering strain and construction method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0087] The microorganisms used in the present invention are wild strains of Rhodotorula viscosus, which are screened and obtained by the inventor from nature. The specific screening method is as follows: the initial screening of bacterial strains uses Bengal red medium (peptone 5g, glucose 10g, potassium dihydrogen phosphate 1g, sulfuric acid Magnesium (MgSO 4 ·7H 2O) 0.5g, agar 20g, 1 / 3000 Bengal red solution 100mL, distilled water 1000mL, chloramphenicol 0.1g. ), YPD medium (1% yeast extract, 2% peptone, 2% glucose) was used for fermentation culture, and the oil production was screened by Sudan black staining method; the candidate strain was further quantitatively determined by hot acid-organic solvent method The oil production is high or low; after two rounds of screening, a strain with high oil production was obtained, and through morphological, physiological and biochemical identification, it was preliminarily judged that it was a strain of yeast; by determining the sequ...

Embodiment 2

[0094] The specific steps of the construction process of Rhodotorula viscosus genetically engineered bacteria are as follows:

[0095] A. Obtaining the target gene:

[0096] (1) For the extraction method of Rhodotorula viscosus genomic DNA, refer to the instruction manual of E.Z.N.A.TM Yeast DNA Kit from Omega Company. The total genomic DNA of Rhodotorula glutinosa was extracted by a modified bead mill combined with phenol-chloroform method. The extracted total DNA was purified and spotted on 1% agarose gel electrophoresis for verification. The genomic template was subpackaged and stored at -20°C, and used for subsequent PCR reactions. Two pairs of primers were designed to amplify 26SrDNA D1 / D2 fragment and 5.8SrDNA-ITS fragment according to the conserved sequence of rDNA in Rhodotorula viscosus genome.

[0097] (2) Using the genome of Mucor circinelloides as a template, degenerate primers were designed according to the homologous and conserved amino acid sequence of malic e...

Embodiment 3

[0111] This example is the application of Rhodotorula glutinis high-yield oil-yielding genetic engineering bacteria (Rhodotorula glutinis) in the production of microbial oil, including two parts: microbial strain fermentation and oil extraction two stages, wherein, the first stage, microbial strain fermentation is Using large-scale high-density nanomaterial concentrated liquid semi-continuous fermentation technology, the nano-material concentrated fermentation liquid high-density semi-continuous fermentation technology large-scale fermentation strains, the biomass and oil production of the strains are about 30 times higher than ordinary fermentation methods, nanomaterials The application of the concentrated solution ensures the full utilization of raw materials and effective oxygen supply. At the same time, the concentration of the fermentation solution reduces the difficulty of subsequent processing, and the semi-continuous fermentation method prolongs the vitality of the strai...

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Abstract

The invention relates to a rhodotorula glutinis oil genetic engineering strain and a construction method and an application thereof. The construction method of the genetic engineering strain is mainly as follows: utilizing rDNA (recombinant deoxyribonucleic acid) of rhodotorula glutinis as a target sequence for homologous integration, using strong promoter genes PGK1 of saccharomyces cerevisiae and malate dehydrogenase genes ME of chaetomium cochloides to construct an expression vector to be introduced into rhodotorula glutinis, and enabling ME genes to obtain high-efficient expression in a rhodotorula glutinis body, wherein the content of lipid in a transformant is improved by 2.5 times in comparison with a wild strain. According to the construction method disclosed by the invention, key enzyme genes and a strong promoter for anabolism of the lipid are introduced on the basis that the anabolism of microbial oil is known, so that the lipid metabolism is regulated and controlled, and the yield of oil is improved. The genetic engineering strain can be applied to production of the microbial oil and development of functional oil related products, such as medicaments, health care products and the like.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a method for constructing a genetic engineering bacterium of Rhodotorula viscosus with high oil production. Background technique [0002] Rhodotorula glutinis (Fres.) Harrison is an important oil-producing microorganism, which can produce microbial oil and other active substances, carotenoids, L-phenylalanine, SOD, etc., and under certain conditions, Produces alpha-alanine and glutamic acid. Due to its simple and extensive nutritional requirements, bagasse, waste molasses, etc. can be used as culture substrates, and the cost is relatively low. In particular, its lipid content is significantly higher than that of other oil-producing microorganisms. In recent years, people have been more and more focused on the development and research of its oil-producing function, and its fermentation technology has been studied, so that the oil production of Rhodotorula viscosus has ob...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N1/16C12P7/64A23L1/30C12R1/645
Inventor 孙晗笑王一杨李智孙晗蓄冯丽霞孙长伟匡超周瑞秒
Owner 广州溯原生物科技股份有限公司
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