Cyclin-Dependent Kinases and CTD Phosphatases in Cell Cycle Transcriptional Control: Conservation across Eukaryotic Kingdoms and Uniqueness to Plants

Cell cycle control is vital for cell proliferation in all eukaryotic organisms. The entire cell cycle can be conceptually separated into four distinct phases, Gap 1 (G1), DNA synthesis (S), G2, and mitosis (M), which progress sequentially. The precise control of transcription, in particular, at the G1 to S and G2 to M transitions, is crucial for the synthesis of many phase-specific proteins, to ensure orderly progression throughout the cell cycle. This mini-review highlights highly conserved transcriptional regulators that are shared in budding yeast (Saccharomyces cerevisiae), Arabidopsis thaliana model plant, and humans, which have been separated for more than a billion years of evolution. These include structurally and/or functionally conserved regulators cyclin-dependent kinases (CDKs), RNA polymerase II C-terminal domain (CTD) phosphatases, and the classical versus shortcut models of Pol II transcriptional control. A few of CDKs and CTD phosphatases counteract to control the Pol II CTD Ser phosphorylation codes and are considered critical regulators of Pol II transcriptional process from initiation to elongation and termination. The functions of plant-unique CDKs and CTD phosphatases in relation to cell division are also briefly summarized. Future studies towards testing a cooperative transcriptional mechanism, which is proposed here and involves sequence-specific transcription factors and the shortcut model of Pol II CTD code modulation, across the three eukaryotic kingdoms will reveal how individual organisms achieve the most productive, large-scale transcription of phase-specific genes required for orderly progression throughout the entire cell cycle.

Study of the Effect of Squalene Epoxidase Activity on Squalene Biosynthesis by Yeast Saccharomyces Cerevisiae VGSh-2

The researchers of this study investigated the biosynthesis of squalene by the yeast S. cerevisiae VGSH-2 through the activity of squalene epoxidase, which is a key enzyme in the conversion of squalene to ergosterol. It has been established that under aerobic conditions the antimycotic drug terbinafine promotes the switching of ergosterol formation to squalene synthesis. This switch occurs through specific inhibition of the squalene epoxidase of the yeast S. cerevisiae VGSH-2, thus increasing the biosynthetic ability of the yeast towards squalene. According to the results of this study, the optimal concentration of terbinofine in the nutrient medium was 0.3 μmol / cm3 . This concentration led to a 5-fold decrease in squalene epoxidase activity and a 7-8 times increase in squalene synthesis. The results obtained can be used to develop a competitive technology for the industrial production of squalene by microbial synthesis. Keywords: squalene, yeast, biosynthesis, inhibition of activity, terbinafine, squalene epoxidase, Saccharomices cerevisiae VGSH-2

Engineered cell differentiation and sexual reproduction in probiotic and mating yeasts

G protein-coupled receptors (GPCRs) enable cells to sense environmental cues and are indispensable for coordinating vital processes including quorum sensing, proliferation, and sexual reproduction. GPCRs comprise the largest class of cell surface receptors in eukaryotes, and for more than three decades the pheromone-induced mating pathway in baker's yeast Saccharomyces cerevisiae has served as a model for studying heterologous GPCRs (hGPCRs). Here we report transcriptome profiles following mating pathway activation in native and hGPCR-signaling yeast, and use a model-guided approach to correlate gene expression to morphological changes. From this we demonstrate mating between haploid cells armed with hGPCRs and endogenous biosynthesis of their cognate ligands. Furthermore, we devise a ligand-free screening strategy for hGPCR compatibility with the yeast mating pathway and enable hGPCR-signaling in the probiotic yeast Saccharomyces boulardii. Combined, our findings enable new means to study mating, hGPCR-signaling, and cell-cell communication in a model eukaryote and yeast probiotics.

Comparison of Different Label-Free Techniques for the Semi-Absolute Quantification of Protein Abundance

In proteomics, it is essential to quantify proteins in absolute terms if we wish to compare results among studies and integrate high-throughput biological data into genome-scale metabolic models. While labeling target peptides with stable isotopes allow protein abundance to be accurately quantified, the utility of this technique is constrained by the low number of quantifiable proteins that it yields. Recently, label-free shotgun proteomics has become the “gold standard” for carrying out global assessments of biological samples containing thousands of proteins. However, this tool must be further improved if we wish to accurately quantify absolute levels of proteins. Here, we used different label-free quantification techniques to estimate absolute protein abundance in the model yeast Saccharomyces cerevisiae. More specifically, we evaluated the performance of seven different quantification methods, based either on spectral counting (SC) or extracted-ion chromatogram (XIC), which were applied to samples from five different proteome backgrounds. We also compared the accuracy and reproducibility of two strategies for transforming relative abundance into absolute abundance: a UPS2-based strategy and the total protein approach (TPA). This study mentions technical challenges related to UPS2 use and proposes ways of addressing them, including utilizing a smaller, more highly optimized amount of UPS2. Overall, three SC-based methods (PAI, SAF, and NSAF) yielded the best results because they struck a good balance between experimental performance and protein quantification.

Yeast Hybrids in Brewing

: Microbiology has long been a keystone in fermentation and the utilization of yeast biology rein-forces molecular biotechnology as the pioneering frontier in brewing science. Consequently, modern understanding of the brewer’s yeast has faced significant refinement over the last few decades. This publication presents a condensed summation of Saccharomyces species dynamics with an emphasis on the relationship between traditional ale yeast, Saccharomyces cerevisiae, and the interspecific hybrids used in lager beer production, S. pastorianus. Introgression from other Sac-charomyces species is also touched on. The unique history of Saccharomyces cerevisiae and Saccharo-myces hybrids are exemplified by recent genomic sequencing studies aimed at categorizing brewing strains through phylogeny and redefining Saccharomyces species boundaries. Phylogenetic investigations highlight the genomic diversity of Saccharomyces cerevisiae ale strains long known to brewers by their fermentation characteristics and phenotypes. Discoveries of genomic contribu-tions from interspecific Saccharomyces species into the genome of S. cerevisiae strains is ever more apparent with increased investigations on the hybrid nature of modern industrial and historical fermentation yeast.

Evaluation of two brazilian native yeast strains (Pichia kudriavzevii) in craft beer

Beer production is an ancient biotechnological process and since yeast as discovered to be responsible for the transformation of barley wort into beer, studies have been carried out with the aim of understanding the behavior of these microorganisms. This work aimed to study the application of two strains of yeasts of the species Pichia kudriavzevii, isolated in the Brazilian Midwest for the production of craft beer and to analyze the occurrence of bioaromatization, with the production of volatile organic compounds (VOC) and to evaluate the sensory perception of the results with untrained end consumers through a quick sensory methodology called Check All That Apply (CATA). For this purpose, three batches of beer were produced and inoculated with commercial yeast (Saccharomyces cerevisiae, the control) and two strains of the same species, called Pichia kudriavzevii BB1 and Pichia kudriavzevii BB2. A total of 28 volatile organic compounds that differentiated the control of the Pichia BB1/BB2 group were detected, being 20 esters, 2 alcohols, 5 carboxylic acids and 1 hydrocarbon. There was no difference among the Pichia kudriavzevii BB1 and BB2 samples (p>0.05) in the sensory analysis using the CATA methodology. It was possible to distinguish two clusters between the tasters according to the habit of consuming craft beer, and those who consumed frequently, assigned a higher score in the hedonic test. It was concluded that Pichia kudriavzevii BB1 and BB2 influenced the beer bioaromatization, improving the acceptance test score with the tasters.

Micro-Bioreactors in Space: Case Study of a Yeast (Saccharomyces cerevisiae) Bioreactor With a Non-Invasive Monitoring Method

Bioreactors in space have applications from basic science to microbial factories. Monitoring bioreactors in microgravity has challenges with respect to fluidics, aeration, sensor size, sample volume and disturbance of medium and cultures. We present a case study of the development of small bioreactors and a non-invasive method to monitor dissolved oxygen, pH, and biomass of yeast cultures. Two different bioreactor configurations were tested for system volumes of 60 ml and 10.5 ml. For both configurations, the PreSens SFR vario, an optical sensor array, collected data autonomously. Oxygen and pH in the cultures were monitored using chemically doped spots, 7 mm in diameter, that were fixed to the bottom of sampling chambers. Spots emitted a fluorescent signal for DO and pH when reacted with oxygen molecules and hydrogen ions, respectively. Biomass was sensed using light reflectance at centered at 605 nm. The, optical array had three light detectors, one for each variable, that returned signals that were pre- and post-calibrated. For heterotrophic cultures requiring oxygen and respiring carbon dioxide, a hollow fiber filter, in-line with the optical array, oxygenated cells and remove carbon dioxide. This provided oxygen levels that were sufficient to maintain aerobic respiration for steady state conditions. Time series of yeast metabolism in the two bioreactors are compared and discussed. The bioreactor configurations can be easily be modified for autotrophic cultures such that carbon dioxide is enhanced and oxygen removed, which would be required for photosynthetic algal cultures.