The current article describes the synthesis, characterization, and application of a designed hybrid fluorescent BTD–coumarin (2,1,3-benzothiadiazole-coumarin) derivative (named BTD-Lip). The use of BTD-Lip for live-cells staining showed excellent results, and lipid droplets (LDs) could be selectively stained. When compared with the commercially available dye (BODIPY) for LD staining, it was noted that the designed hybrid fluorescence was capable of staining a considerable larger number of LDs in both live and fixed cells (ca. 40% more). The new dye was also tested on live Caenorhabditis elegans (complex model) and showed an impressive selectivity inside the worm, whereas the commercial dye showed no selectivity in the complex model.

Biosurfactants are biological tensioactive agents that can be used in the cosmetic and food industries. Rhamnolipids are glycolipid biosurfactants naturally produced by Pseudomonas aeruginosa and are composed of one or two rhamnose molecules linked to beta-hydroxy fatty acid chains. These compounds are green alternatives to petrochemical surfactants, but their large-scale production is still in its infancy, hindered due to pathogenicity of natural producer, high substrate and purification costs and low yields and productivities. This study, for the first time, aimed at producing mono-rhamnolipids from sucrose by recombinant GRAS Saccharomyces cerevisiae strains. Six enzymes from P. aeruginosa involved in mono-rhamnolipid biosynthesis were functionally expressed in the yeast. Furthermore, its SUC2 invertase gene was disrupted and a sucrose phosphorylase gene from Pelomonas saccharophila was also expressed to reduce the pathway’s overall energy requirement. Two strains were constructed aiming to produce mono-rhamnolipids and the pathway’s intermediate dTDP-L-rhamnose. Production of both molecules was analyzed by confocal microscopy and mass spectrometry, respectively. These strains displayed, for the first time as a proof of concept, the potential of production of these molecules by a GRAS eukaryotic microorganism from an inexpensive substrate. These constructs show the potential to further improve rhamnolipids production in a yeast-based industrial bioprocess.

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A fluorescent 2,1,3-benzothiadiazole-containing goniothalamin derivative, BTD−GTN (1), has been synthesized and successfully tested in bioimaging experiments in live cells. The fluorescent compound proved to be capable of transposing the cell membranes, indicating its subcellular localization. The use of the benzothiadiazole core as the fluorophore revealed the favored localization of the GTN analogue 1 in the cytoplasm of live cells, preferentially in the mitochondria, in line with previous results that indicated the loss of mitochondrial transmembrane potential upon treatment with GTN. The results described herein highlight the potential of the BTD–GTN hybrid structures for future studies regarding the cellular mechanism of action of this family of compounds.

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