scholarly journals Nile Red Incubation Time Before Reading Fluorescence Greatly Influences the Yeast Neutral Lipids Quantification

2021 ◽  
Vol 12 ◽  
Author(s):  
Mauricio Ramírez-Castrillón ◽  
Victoria P. Jaramillo-Garcia ◽  
Helio Lopes Barros ◽  
João A. Pegas Henriques ◽  
Valter Stefani ◽  
...  
Keyword(s):  
Lipid Content ◽  
High Throughput Screening ◽  
Oleaginous Yeast ◽  
Neutral Lipids ◽  
Nile Red ◽  
Oleaginous Yeasts ◽  
Red Fluorescence ◽  
Fluorescence Measurements ◽  
Yeast Strains ◽  
Nile Red Fluorescence

High-throughput screening methodologies to estimate lipid content in oleaginous yeasts use Nile red fluorescence in a given solvent and optimized excitation/emission wavelengths. However, Nile red fluorescence stabilization has been poorly analyzed, and high variability occurs when relative fluorescence is measured immediately or a few minutes after dye addition. The aim of this work was to analyze the fluorescence of Nile red at different incubation times using a variety of solvents and oleaginous/non-oleaginous yeast strains. We showed that fluorescence stabilization occurs between 20 and 30 min, depending on the strain and solvent. Therefore, we suggest that fluorescence measurements should be followed until stabilization, where Relative Fluorescence Units should be considered after stabilization for lipid content estimation.

scholarly journals Dataset of Nile Red Fluorescence Readings with Different Yeast Strains, Solvents, and Incubation Times

Data ◽  
2020 ◽  
Vol 5 (3) ◽  
pp. 77
Author(s):  
Mauricio Ramirez-Castrillon ◽  
Victoria Jaramillo-Garcia ◽  
Helio Barros ◽  
João Henriques ◽  
Valter Stefani ◽  
...  
Keyword(s):  
Culture Medium ◽  
Isopropyl Alcohol ◽  
Oleaginous Yeast ◽  
Nile Red ◽  
Solvent System ◽  
Oleaginous Yeasts ◽  
Yeast Strains ◽  
Nile Red Fluorescence ◽  
Accumulation Kinetics ◽  
Red Dye

We used Nile red to estimate lipid content in oleaginous yeasts using a high-throughput approach. We measured the fluorescence intensity of Nile red using different solvents, yeast strains, and incubation times in optimized excitation/emission wavelengths. The data show the relative fluorescence units (RFU) for Nile red excitation, using 1× PBS, 1× PBS and 5% v/v isopropyl alcohol, 50% v/v glycerol, culture medium A-gly broth, and A-gly broth supplemented with 5% v/v DMSO. In addition, we showed the RFU for the Nile red dye for different oleaginous and non-oleaginous yeast strains, such as Meyerozyma guilliermondii BI281A, Yarrowia lipolytica QU21 and Saccharomyces cerevisiae MRC164. Other measurements of lipid accumulation kinetics were shown for the above and additional yeast strains. These datasets provide the guidelines to obtain the optimal solvent system and the minimal interaction time for the Nile red dye to enter in the cells and obtain a stable readout.

scholarly journals Modified high-throughput Nile red fluorescence assay for the rapid screening of oleaginous yeasts using acetic acid as carbon source

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Catarina Miranda ◽  
Sara Bettencourt ◽  
Tatiana Pozdniakova ◽  
Joana Pereira ◽  
Paula Sampaio ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Carbon Source ◽  
High Throughput ◽  
Nile Red ◽  
Fluorescence Assay ◽  
Rapid Screening ◽  
Oleaginous Yeasts ◽  
Red Fluorescence ◽  
Nile Red Fluorescence

Directed evolution of a bacterial WS/DGAT acyltransferase: improving tDGAT from Thermomonospora curvata

2019 ◽  
Author(s):  
Omar Santín ◽  
Serena Galié ◽  
Gabriel Moncalián
Keyword(s):  
Directed Evolution ◽  
High Throughput Screening ◽  
Neutral Lipids ◽  
Nile Red ◽  
Diacylglycerol Acyltransferase ◽  
Wax Ester ◽  
Bacterial Lipid ◽  
Acyl Coenzyme A ◽  
Wax Ester Synthase ◽  
Tag Accumulation

Abstract Some bacteria belonging to the actinobacteria and proteobacteria groups can accumulate neutral lipids expressing enzymes of the wax ester synthase/acyl coenzyme A: diacylglycerol acyltransferase (WS/DGAT) family. tDGAT is a WS/DGAT-like enzyme from Thermomonospora curvata able to produce TAGs and WEs when heterologously expressed in Escherichia coli. In this study, a protocol for the directed evolution of bacterial lipid-producing enzymes based on fluorimetry is developed and tested. tDGAT has been successfully evolved towards the improvement of TAG production with an up to 2.5 times increase in TAG accumulation. Mutants with no ability to produce TAGs but able to accumulate waxes were also selected during the screening. The localization of the mutations that enhance TAG production in the outer surface of tDGAT points out possible new mechanisms that contribute to the activity of this family of enzymes. This Nile red-based high throughput screening provides an evolution platform for other WS/DGAT-like enzymes.

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