A fast method to distinguish between fermentative and respiratory metabolisms in single yeast cells.

Saccharomyces cerevisiae adjusts its metabolism based on nutrient availability, typically transitioning from glucose fermentation to ethanol respiration as glucose becomes limiting. However, our understanding of the regulation of metabolism is largely based on population averages, whereas nutrient transitions may cause heterogeneous responses. Here we introduce iCRAFT, a method that couples the ATP Förster resonance energy transfer (FRET)-based biosensor yAT1.03 with Antimycin A to differentiate fermentative and respiratory metabolisms in individual yeast cells. Upon Antimycin A addition, respiratory cells experienced a sharp decrease of the normalized FRET ratio, while respiro-fermentative cells showed no response. Next, we tracked changes in metabolism during the diauxic shift of a glucose pre-grown culture. Following glucose exhaustion, the entire cell population experienced a progressive rise in cytosolic ATP produced via respiration, suggesting a gradual increase in respiratory capacity. Overall, iCRAFT is a robust tool to distinguish fermentation from respiration, offering a new single-cell opportunity to study yeast metabolism.

Ethanol-lactate transition of Lachancea thermotolerans is linked to nitrogen metabolism.

Climate change increases sugar content in grapes, resulting in unwanted increase in ethanol content of wine. Lachancea thermotolerans ferments glucose and fructose into both ethanol and lactate, decreasing final ethanol content and positively affecting wine acidity. Reported Lachancea thermotolerans strains show big variation in lactate production during fermentation. However, a mechanistic understanding of this lactate producing phenotype is currently lacking. Through a combination of metabolomics, transcriptomics, genomics and computational methods we show that the lactate production is induced by amino acid limitation in a high lactate producing strain. We found in fermentations in synthetic grape juice media that lactate production starts in the last stages of growth, marked by decreased growth rate and increased expression levels of stress related genes. This onset of lactate production is specific for the high lactate producing strain and independent of oxygen availability. The onset of lactate production was changed by increased amino acid content of the media, and it is shown by both computational methods and amino acid measurements that at the onset of lactate production amino acids become limiting for growth. This study shows that lactate production of Lachancea thermotolerans is directly linked to nitrogen availability in the media, an insight that can further aid in the improvement of wine quality.

Engineering the thermotolerant industrial yeast Kluyveromyces marxianus for anaerobic growth.

Current large-scale, anaerobic industrial processes for ethanol production from renewable carbohydrates predominantly rely on the mesophilic yeast Saccharomyces cerevisiae. Use of thermotolerant, facultatively fermentative yeasts such as Kluyveromyces marxianus could confer significant economic benefits. However, in contrast to S. cerevisiae, these yeasts cannot grow in the absence of oxygen. Responses of K. marxianus and S. cerevisiae to different oxygen-limitation regimes were analyzed in chemostats. Genome and transcriptome analysis, physiological responses to sterol supplementation and sterol-uptake measurements identified absence of a functional sterol-uptake mechanism as a key factor underlying the oxygen requirement of K. marxianus. Heterologous expression of a squalene-tetrahymanol cyclase enabled oxygen-independent synthesis of the sterol surrogate tetrahymanol in K. marxianus. After a brief adaptation under oxygen-limited conditions, tetrahymanol-expressing K. marxianus strains grew anaerobically on glucose at temperatures of up to 45 °C. These results open up new directions in the development of thermotolerant yeast strains for anaerobic industrial applications.

Quantitation of DNA-Binding Affinity Using Tethered Particle Motion.

The binding constant is an important characteristic of a DNA-binding protein. A large number of methods exist to measure the binding constant, but many of those methods have intrinsic flaws that influence the outcome of the characterization. Tethered Particle Motion (TPM) is a simple, cheap, and high-throughput single-molecule method that can be used to reliably measure binding constants of proteins binding to DNA, provided that they distort DNA. In TPM, the motion of a bead tethered to a surface by DNA is tracked using light microscopy. A protein binding to the DNA will alter bead motion. This makes it possible to measure binding properties. We use the bacterial protein Integration Host Factor (IHF) as an example to show how specific binding to DNA can be measured. Moreover, we show a new intuitive quantitative approach to displaying data obtained via TPM.