Oil-resistant saccharomyces cerevisiae genetically engineered bacterium and construction method thereof

A technology of genetically engineered bacteria and Saccharomyces cerevisiae, which is applied in the field of oil-resistant Saccharomyces cerevisiae genetically engineered bacteria and its construction, can solve the problem that the activity cannot completely break through the limitations of cell lipid synthesis and storage, and achieve improved cell tolerance and intracellular lipid The effect of qualitative component enhancement

Pending Publication Date: 2022-03-01
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the limited space in the cell, simply increasing the activity of key rate-limiting enzymes cannot completely break through the limitation of cellular lipid synthesis and storage

Method used

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  • Oil-resistant saccharomyces cerevisiae genetically engineered bacterium and construction method thereof
  • Oil-resistant saccharomyces cerevisiae genetically engineered bacterium and construction method thereof
  • Oil-resistant saccharomyces cerevisiae genetically engineered bacterium and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Construction of oil-resistant Saccharomyces cerevisiae strains

[0032] 1.1 Construction of SEI1::Are2 gene replacement module

[0033] (1) Using the genomic DNA of the original Saccharomyces cerevisiae BY4741 and the pUG6 plasmid as a template, the upstream of the SEI1 gene, the G418 resistance gene, the GAP promoter, the Are2 gene, the CYC1 terminator, and the downstream homology arm of the SEI1 gene were amplified by common PCR amplification. PCR reaction system is shown in Table 1. PCR reaction conditions are as follows: 1) Pre-denaturation at 98°C for 5 minutes; 2) Denaturation at 98°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 2 minutes, three steps for 30 cycles, 72°C Extend for another 10 min. Among them, the annealing temperature depends on the Tm value of the primer, and the extension time at 72°C depends on the length of the amplified fragment (1kb·min -1 ). The above gene fragments were obtained by agarose gel elect...

Embodiment 2

[0055] Example 2: Comparison of Saccharomyces cerevisiae cell viability under oleic acid-induced stress

[0056] Pick a single colony of wild-type BY4741 and modified bacteria, inoculate it in YPO seed solution and cultivate it for 24 hours, collect the cells, and dilute the fine concentration to OD with YPO medium 600 After = 0.1, the starting bacteria and the modified strains were respectively inoculated in 96-well plates (200 μL / well) and cultured for 72 hours. Three replicate wells were made for each group of experiments, and the cell viability of tetrazolium salt (MTT) was detected every 24 hours during this period.

[0057] The above YPO medium formula is as follows: 0.3% yeast powder, 0.5 peptone, 0.6% glucose, 0.5% KH 2 PO 2 , 0.2% oleic acid, and add 0.2% tween-80 to dissolve the oleic acid.

[0058] Quantitative analysis of MTT cell viability. After culturing at 30°C for 24, 48, and 72 hours, the cell viability of the original strain BY4741 and the modified strain...

Embodiment 3

[0060] Example 3: Nile Red Stained Cell Lipid Droplet Structure and Lipid Content Comparison

[0061] The present invention adopts the Nile red staining method to stain the lipid droplet structure of the cells. Collect BY4741 and BY4741-SEI1::Are2 / Tgl1::Yft2 lipid-tolerant cells cultured in YPD medium at 30°C for 24 hours, and take 1 mL of bacterial liquid for staining experiment. Dissolve Nile Red in DMSO, then add it to the post-incubation solution, keep its final concentration at 2 μg / ml, and incubate at 30°C for 20 min. Pipette 5 μL of the incubated mixed solution and drop it on a glass slide, place it under a fluorescent electron microscope, and use the optical channel and the RFP fluorescent cube channel (Ex=531 / 40; Em=593 / 40) for observation. Oval or spherical Saccharomyces cerevisiae cells with a width of 2.5-5 μm and a length of 4.5-10 μm were observed in the 100× field of view, as well as lipid droplets with a diameter of about 1 μm and yellow fluorescence in the ce...

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Abstract

The invention discloses an oil-resistant saccharomyces cerevisiae genetically engineered bacterium and a construction method thereof, and belongs to the technical field of bioengineering. The invention discloses a saccharomyces cerevisiae genetically engineered bacterium capable of overexpressing Are2 and Yft2 genes and replacing SEI1 and Tgl1 genes at the same time. The saccharomyces cerevisiae is modified by replacing genes with original saccharomyces cerevisiae by using a homologous recombination technology. The amount of sterol substances stored in the original strain BY4741 is limited, and excessive accumulated lipid intermediates can generate a product inhibition effect, so that synthesis of a target product is directly influenced, cytotoxicity can be caused, and cell viability can be reduced. Compared with an original starting strain, the modified oil-resistant saccharomyces cerevisiae genetically engineered bacterium has the advantages that the volume ratio of a lipid droplet structure of a cell lipid storage unit is remarkably increased, the cytotoxicity and product inhibition caused by lipid accumulation are effectively reduced, and the intracellular cumulant of ester compounds is remarkably increased; and a new strategy and a good tolerant cell model are provided for breaking through the de novo synthesis limit of the ester compound saccharomyces cerevisiae.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to an oil-resistant Saccharomyces cerevisiae genetically engineered bacterium and a construction method thereof. Background technique [0002] Lipids are diverse and ubiquitous compounds, such as fatty acids, phospholipids, sterols, sphingolipids, terpenes, etc., have many key biological functions, and participate in the structural composition of cell membranes, energy storage, and signal transduction processes , is a representative product of medicine, food and bulk chemicals. [0003] Saccharomyces cerevisiae has the advantages of relatively complete genetic manipulation, high availability of molecular information, short passage time, easy culture and high tolerance to organic solvents, and is often used as an important chassis for the production of high-value drugs through metabolic engineering. By strengthening the fatty acid synthesis pathway, the efficient ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12N15/31C12R1/865
CPCC07K14/395C12N15/81
Inventor 柳志强曹丽莎沈逸柯霞郑裕国
Owner ZHEJIANG UNIV OF TECH
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