Yeast “Make-Accumulate-Consume” Life Strategy Evolved as a Multi-Step Process That Predates the Whole Genome Duplication

When fruits ripen, microbial communities start a fierce competition for the freely available fruit sugars. Three yeast lineages, including baker’s yeast Saccharomyces cerevisiae, have independently developed the metabolic activity to convert simple sugars into ethanol even under fully aerobic conditions. This fermentation capacity, named Crabtree effect, reduces the cell-biomass production but provides in nature a tool to out-compete other microorganisms. Here, we analyzed over forty Saccharomycetaceae yeasts, covering over 200 million years of the evolutionary history, for their carbon metabolism. The experiments were done under strictly controlled and uniform conditions, which has not been done before. We show that the origin of Crabtree effect in Saccharomycetaceae predates the whole genome duplication and became a settled metabolic trait after the split of the S. cerevisiae and Kluyveromyces lineages, and coincided with the origin of modern fruit bearing plants. Our results suggest that ethanol fermentation evolved progressively, involving several successive molecular events that have gradually remodeled the yeast carbon metabolism. While some of the final evolutionary events, like gene duplications of glucose transporters and glycolytic enzymes, have been deduced, the earliest molecular events initiating Crabtree effect are still to be determined.     

The Effect of Attractive Interactions and Macromolecular Crowding on Crystallins Association

In living systems proteins are typically found in crowded environments where their effective interactions strongly depend on the surrounding medium. Yet, their association and dissociation needs to be robustly controlled in order to enable biological function. Uncontrolled protein aggregation often causes disease. For instance, cataract is caused by the clustering of lens proteins, i.e., crystallins, resulting in enhanced light scattering and impaired vision or blindness. To investigate the molecular origins of cataract formation and to design efficient treatments, a better understanding of crystallin association in macromolecular crowded environment is needed. Here we present a theoretical study of simple coarse grained colloidal models to characterize the general features of how the association equilibrium of proteins depends on the magnitude of intermolecular attraction. By comparing the analytic results to the available experimental data on the osmotic pressure in crystallin solutions, we identify the effective parameters regimes applicable to crystallins. Moreover, the combination of two models allows us to predict that the number of binding sites on crystallin is small, i.e. one to three per protein, which is different from previous estimates. We further observe that the crowding factor is sensitive to the size asymmetry between the reactants and crowding agents, the shape of the protein clusters, and to small variations of intermolecular attraction. Our work may provide general guidelines on how to steer the protein interactions in order to control their association.     

Continuous Solvent/Detergent Virus Inactivation Using a Packed‐Bed Reactor

Continuous virus inactivation (VI) remains one of the missing pieces while the biopharma industry moves toward continuous manufacturing. The challenges of adapting VI to the continuous operation are two‐fold: 1) achieving fluid homogeneity and 2) a narrow residence time distribution (RTD) for fluid incubation. To address these challenges, a dynamic active in‐line mixer and a packed‐bed continuous virus inactivation reactor (CVIR) are implemented, which act as a narrow RTD incubation chamber. The developed concept is applied using solvent/detergent (S/D) treatment for inactivation of two commonly used model viruses. The in‐line mixer is characterized and enables mixing of the viscous S/D chemicals to ±1.0% of the target concentration in a small dead volume. The reactor's RTD is characterized and additional control experiments confirm that the VI is due to the S/D action and not induced by system components. The CVIR setup achieves steady state rapidly before two reactor volumes and the logarithmic reduction values of the continuous inactivation process are identical to those obtained by the traditional batch operation. The packed‐bed reactor for continuous VI unites fully continuous processing with very low‐pressure drop and scalability.     

Kinetic model of ethopropazine interaction with horse serum butyrylcholinesterase and its docking into the active site.

The action of a potent tricyclic cholinesterase inhibitor ethopropazine on the hydrolysis of acetylthiocholine and butyrylthiocholine by purified horse serum butyrylcholinesterase (EC 3.1.1.8) was investigated at 25 and 37 degrees C. The enzyme activities were measured on a stopped-flow apparatus and the analysis of experimental data was done by applying a six-parameter model for substrate hydrolysis. The model, which was introduced to explain the kinetics of Drosophila melanogaster acetylcholinesterase [Stojan et al. (1998) FEBS Lett. 440, 85-88], is defined with two dissociation constants and four rate constants and can describe both cooperative phenomena, apparent activation at low substrate concentrations and substrate inhibition by excess of substrate. For the analysis of the data in the presence of ethopropazine at two temperatures, we have enlarged the reaction scheme to allow primarily its competition with the substrate at the peripheral site, but the competition at the acylation site was not excluded. The proposed reaction scheme revealed, upon analysis, competitive effects of ethopropazine at both sites; at 25 degrees C, three enzyme-inhibitor dissociation constants could be evaluated; at 37 degrees C, only two constants could be evaluated. Although the model considers both cooperative phenomena, it appears that decreased enzyme sensitivity at higher temperature, predominantly for the ligands at the peripheral binding site, makes the determination of some expected enzyme substrate and/or inhibitor complexes technically impossible. The same reason might also account for one of the paradoxes in cholinesterases: activities at 25 degrees C at low substrate concentrations are higher than at 37 degrees C. Positioning of ethopropazine in the active-site gorge by molecular dynamics simulations shows that A328, W82, D70, and Y332 amino acid residues stabilize binding of the inhibitor.     

Plant dihydroorotate dehydrogenase differs significantly in substrate specificity and inhibition from the animal enzymes

The mitochondrial membrane bound dihydroorotate dehydrogenase (DHODH; EC 1.3.99.11) catalyzes the fourth step of pyrimidine biosynthesis. By the present correction of a known cDNA sequence for Arabidopsis thaliana DHODH we revealed the importance of the very C-terminal part for its catalytic activity and the reason why--in contrast to mammalian and insect species--the recombinant plant flavoenzyme was unaccessible to date for in vitro characterization. Structure-activity relationship studies explained that potent inhibitors of animal DHODH do not significantly affect the plant enzyme. These difference could be exploited for a novel approach to herb or pest growth control by limitation of pyrimidine nucleotide pools.     

Multi-step analysis as a tool for kinetic parameter estimation and mechanism discrimination in the reaction between tight-binding fasciculin 2 and electric eel acetylcholinesterase

The mechanism of action of a potent peptidic inhibitor fasciculin 2 (Fas2) on electric eel acetylcholinesterase (eleelAChE) has been examined in a three-level analysis. Classical steps included equilibration experiments for the evaluation of high affinity binding constant and the existence of residual hydrolytic activity in a solution of completely Fas2 saturated enzyme. The two rate constants for the association (k(on)) and the dissociation (k(off)) of Fas2 with free enzyme were determined by the time course of residual enzyme activity measurements. In the third step, with a nonclassical progress curve analysis, we found that the Fas2-enzyme complex exhibited hydrolytic activity in a butyrylcholinesterase-like kinetics. The switch appears to be a consequence of steric obstruction, but also the consequence of subtle rapid conformational changes around catalytic site, upon slow single-step binding of large Fas2 molecule at the peripheral site. An unusual unilateral effect of bound Fas2 is reflected by acylation-independent association and dissociation rates and might indeed be due to inability of small acylation agent to influence the binding of a large opponent.     

Pleurotus and Agrocybe hemolysins,new proteins hypothetically involved in fungal fruiting

Novel hemolytic proteins, ostreolysin and aegerolysin, were purified from the fruiting bodies of the edible mushrooms Pleurotus ostreatus and Agrocybe aegerita. Both ostreolysin and aegerolysin have a molecular weight of about 16 kDa, have low isoelectric points of 5.0 and 4.85, are thermolabile, and hemolytic to bovine erythrocytes at nanomolar concentrations. Their activity is impaired by micromolar Hg(2+) but not by membrane lipids and serum low-density lipoproteins (LDL). The sequence of respectively 50 and 10 N-terminal amino acid residues of ostreolysin and aegerolysin has been determined and found to be highly identical with a cDNA-derived amino acid sequence of putative Aa-Pri1 protein from the mushroom A. aegerita, Asp-hemolysin from Aspergillus fumigatus, and two bacterial hemolysin-like proteins expressed during sporulation. We found that ostreolysin is expressed during formation of primordia and fruiting bodies, which is in accord with previous finding that the Aa-Pri1 gene is specifically expressed during fruiting initiation. It is suggestive that the isolated hemolysins play an important role in initial phase of fungal fruiting.     

A Study on the Fundamental Mechanism and the Evolutionary Driving Forces behind Aerobic Fermentation in Yeast

Baker’s yeast Saccharomyces cerevisiae rapidly converts sugars to ethanol and carbon dioxide at both anaerobic and aerobic conditions. The later phenomenon is called Crabtree effect and has been described in two forms, long-term and short-term effect. We have previously studied under fully controlled aerobic conditions forty yeast species for their central carbon metabolism and the presence of long-term Crabtree effect. We have also studied ten steady-state yeast cultures, pulsed them with glucose, and followed the central carbon metabolism and the appearance of ethanol at dynamic conditions. In this paper we analyzed those wet laboratory data to elucidate possible mechanisms that determine the fate of glucose in different yeast species that cover approximately 250 million years of evolutionary history. We determine overflow metabolism to be the fundamental mechanism behind both long- and short-term Crabtree effect, which originated approximately 125–150 million years ago in the Saccharomyces lineage. The “invention” of overflow metabolism was the first step in the evolution of aerobic fermentation in yeast. It provides a general strategy to increase energy production rates, which we show is positively correlated to growth. The “invention” of overflow has also simultaneously enabled rapid glucose consumption in yeast, which is a trait that could have been selected for, to “starve” competitors in nature. We also show that glucose repression of respiration is confined mainly among S. cerevisiae and closely related species that diverged after the whole genome duplication event, less than 100 million years ago. Thus, glucose repression of respiration was apparently “invented” as a second step to further increase overflow and ethanol production, to inhibit growth of other microbes. The driving force behind the initial evolutionary steps was most likely competition with other microbes to faster consume and convert sugar into biomass, in niches that were semi-anaerobic.     

Rational design of novel mutants of fungal 17beta-hydroxysteroid dehydrogenase

Reduction of 17-ketosteroids is a biocatalytic process of economic significance for the production of steroid drugs. This reaction can be catalyzed by different microbial 17beta-hydroxysteroid dehydrogenases (17beta-HSD), like the 17beta-HSD activity of Saccharomyces cerevisiae, Pichia faranosa and Mycobacterium sp., and by purified 3beta,17beta-HSD from Pseudomonas testosteroni. In addition to the bacterial 3beta,17beta-HSD the 17beta-HSD of the filamentous fungus Cochliobolus lunatus is the only microbial 17beta-HSD that has been expressed as a recombinant protein and fully characterized. On the basis of its modeled 3D structure, we selected several positions for the replacement of amino acids by site-directed mutagenesis to change substrate specificity, alter coenzyme requirements, and improve overall catalytic activity. Replacement of Val161 and Tyr212 in the substrate-binding region by Gly and Ala, respectively, increased the initial rates for the conversion of androstenedione to testosterone. Replacement of Tyr49 within the coenzyme binding site by Asp changed the coenzyme specificity of the enzyme. This latter mutant can convert the steroids not only in the presence of NADP(+) and NADPH, but also in the presence of NADH and NAD(+). The replacement of His164, located in the non-flexible part of the 'lid' covering the active center resulted in a conformation of the enzyme that possessed a higher catalytic activity.