Microbial cell autofluorescence as a method for measuring the intracellular content of B2 and B6 vitamins.

Vitamins are important organic compound required for the proper functioning of cells and organisms. Vitamins of special industrial and pharmaceutical interests include riboflavin (vitamin B2) and pyridoxine (vitamin B6). Commercial production of those biological compounds has increasingly relied on microorganisms and requires simple methods for detecting and estimating their level of synthesis during the biotechnological process. In the case of yeast, methods based on autofluorescence, i.e. natural fluorescence emitted by several cellular compounds, including vitamins, may be useful. Considering that the intensity of emitted light is proportional to the intracellular concentration of riboflavin and pyridoxine, autofluorescence may be a convenient method for their quantification. In this report, we demonstrate a simple, rapid, and sufficiently trustworthy spectrofluorimetric method for determining the content of vitamins B2 and B6 in yeast cells which consists of cells growing, harvesting, washing, and resuspending in a buffer, and then measuring the emitted visible light using specific wavelength of excitation (λex=340 nm and λem=385 nm for pyridoxine; λex=460 nm and λem=535 nm for riboflavin). The limits of detection (LOD) and quantification (LOQ) estimated through measurements of vitamin fluorescence were below 0.005 µg/ml for riboflavin and below 0.05 µg/ml for pyridoxine, respectively. In turn, the smallest credible cell density for measuring autofluorescence was set at 1×108 yeast cells/ml. The relative level of the cell's autofluorescence can be expressed in mass units by applying proper calculation formulas. A comparison of the autofluorescence-based method with the reference HPLC-UV method shows that autofluorescence measurement can be used in the screening analysis of vitamin content (especially riboflavin) in microbial cells.

Redox perturbations in yeast cells lacking glutathione reductase.

Cellular redox homeostasis has a major effect on cell functions and its maintenance is supported by glutathione and protein thiols which serve as redox buffers in cells. The regulation of the glutathione biosynthetic pathway is a focus of a lot of scientific research. However, still little is known about how complex cellular networks influence glutathione homeostasis. In this work was used an experimental system based on an S. cerevisiae yeast mutant with a lack of the glutathione reductase enzyme and allyl alcohol as a precursor of acrolein inside the cell to determine the cellular processes influencing glutathione homeostasis. The absence of Glr1p slows down the growth rate of the cell population, especially in the presence of allyl alcohol, but does not lead to complete inhibition of the cell's reproductive capacity. It also amends the GSH/GSSG ratio and the share of NADPH and NADP+ in the total NADP(H) pool. The obtained results show that potential pathways involved in the maintenance of redox homeostasis are based from one side on de novo synthesis of GSH as indicated by increased activity of γ-GCS and increased expression of GSH1 gene in the Δglr1 mutant, from the other hand, on increased the level of NADPH. This is because the lower ratio of GSH/GSSG can be counterbalanced with the NADPH/NADP+ alternative system. The higher level of NADPH can be used by the thioredoxin system and other enzymes requiring NADPH to reduce cytosolic GSSG and maintain glutathione redox potential.