Physiological relevance, localization and substrate specificity of the alternative (type II) mitochondrial NADH dehydrogenases of .

Mitochondria from harbor a branched electron-transport chain containing a proton-pumping Complex I NADH dehydrogenase and three Type II NADH dehydrogenases (NDH-2). To investigate the physiological role, localization and substrate specificity of these enzymes, the growth of various NADH dehydrogenase knockout mutants was quantitatively characterized in shake-flask and chemostat cultures, followed by oxygen-uptake experiments with isolated mitochondria. NAD(P)H:quinone oxidoreduction of the three NDH-2 were individually assessed.

Specific growth rates and growth stoichiometries of Saccharomycotina yeasts on ethanol as sole carbon and energy substrate.

Emerging low-emission production technologies make ethanol an interesting substrate for yeast biotechnology, but information on growth rates and biomass yields of yeasts on ethanol is scarce. Strains of 52 Saccharomycotina yeasts were screened for growth on ethanol. The 21 fastest strains, among which representatives of the Phaffomycetales order were overrepresented, showed specific growth rates in ethanol-grown shake-flask cultures between 0.

Optimizing the balance between heterologous acetate- and CO2-reduction pathways in anaerobic cultures of Saccharomyces cerevisiae strains engineered for low-glycerol production.

In anaerobic Saccharomyces cerevisiae cultures, NADH (reduced form of nicotinamide adenine dinucleotide)-cofactor balancing by glycerol formation constrains ethanol yields. Introduction of an acetate-to-ethanol reduction pathway based on heterologous acetylating acetaldehyde dehydrogenase (A-ALD) can replace glycerol formation as 'redox-sink' and improve ethanol yields in acetate-containing media. Acetate concentrations in feedstock for first-generation bioethanol production are, however, insufficient to completely replace glycerol formation.

Co-cultivation of Saccharomyces cerevisiae strains combines advantages of different metabolic engineering strategies for improved ethanol yield.

Glycerol is the major organic byproduct of industrial ethanol production with the yeast Saccharomyces cerevisiae. Improved ethanol yields have been achieved with engineered S. cerevisiae strains in which heterologous pathways replace glycerol formation as the predominant mechanism for anaerobic re-oxidation of surplus NADH generated in biosynthetic reactions.

Quantification and mitigation of byproduct formation by low-glycerol-producing Saccharomyces cerevisiae strains containing Calvin-cycle enzymes.

Background: Anaerobic Saccharomyces cerevisiae cultures require glycerol formation to re-oxidize NADH formed in biosynthetic processes. Introduction of the Calvin-cycle enzymes phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) has been shown to couple re-oxidation of biosynthetic NADH to ethanol production and improve ethanol yield on sugar in fast-growing batch cultures. Since growth rates in industrial ethanol production processes are not constant, performance of engineered strains was studied in slow-growing cultures.

An engineered non-oxidative glycolytic bypass based on Calvin-cycle enzymes enables anaerobic co-fermentation of glucose and sorbitol by Saccharomyces cerevisiae.

Background: Saccharomyces cerevisiae is intensively used for industrial ethanol production. Its native fermentation pathway enables a maximum product yield of 2 mol of ethanol per mole of glucose. Based on conservation laws, supply of additional electrons could support even higher ethanol yields.

Interpretable Identification of Comorbidities Associated with Recurrent ED and Inpatient Visits.

In the hospital setting, a small percentage of recurrent frequent patients contribute to a disproportional amount of healthcare resource utilization. Moreover, in many of these cases, patient outcomes can be greatly improved by reducing re-occurring visits, especially when they are associated with substance abuse, mental health, and medical factors that could be improved by social-behavioral interventions, outpatient or preventative care. Additionally, health care costs can be reduced significantly with fewer preventable recurrent visits.

Automated Evolutionary Engineering of Yeasts.

Evolutionary engineering of microbes provides a powerful tool for untargeted optimization of (engineered) cell factories and identification of genetic targets for further research. Directed evolution is an intrinsically time-intensive effort, and automated methods can significantly reduce manual labor. Here, design considerations for various evolutionary engineering methods are described, and generic workflows for batch-, chemostat-, and accelerostat-based evolution in automated bioreactors are provided.

Carbon dioxide fixation via production of succinic acid from glycerol in engineered Saccharomyces cerevisiae.

Background: The microbial production of succinic acid (SA) from renewable carbon sources via the reverse TCA (rTCA) pathway is a process potentially accompanied by net-fixation of carbon dioxide (CO). Among reduced carbon sources, glycerol is particularly attractive since it allows a nearly twofold higher CO-fixation yield compared to sugars. Recently, we described an engineered Saccharomyces cerevisiae strain which allowed SA production in synthetic glycerol medium with a maximum yield of 0.

Engineering proton-coupled hexose uptake in Saccharomyces cerevisiae for improved ethanol yield.

Background: In the yeast Saccharomyces cerevisiae, which is widely applied for industrial bioethanol production, uptake of hexoses is mediated by transporters with a facilitated diffusion mechanism. In anaerobic cultures, a higher ethanol yield can be achieved when transport of hexoses is proton-coupled, because of the lower net ATP yield of sugar dissimilation. In this study, the facilitated diffusion transport system for hexose sugars of S.

Towards closed carbon loop fermentations: Cofeeding of Yarrowia lipolytica with glucose and formic acid.

A novel fermentation process was developed in which renewable electricity is indirectly used as an energy source in fermentation, synergistically decreasing both the consumption of sugar as a first generation carbon source and emission of the greenhouse gas CO . As an illustration, a glucose-based process is co-fed with formic acid, which can be generated by capturing CO from fermentation offgas followed by electrochemical reduction with renewable electricity. This "closed carbon loop" concept is demonstrated by a case study in which cofeeding formic acid is shown to significantly increase the yield of biomass on glucose of the industrially relevant yeast species Yarrowia lipolytica.

Novel Evolutionary Engineering Approach to Alter Substrate Specificity of Disaccharide Transporter Mal11 in .

A major challenge in the research of transport proteins is to understand how single amino acid residues contribute to their structure and biological function. Amino acid substitutions that result in a selective advantage in adaptive laboratory evolution experiments can provide valuable hints at their role in transport proteins. In this study, we applied an evolutionary engineering strategy to alter the substrate specificity of the proton-coupled disaccharide transporter Mal11 in , which has affinity for sucrose, maltose and glucose.

Respiratory reoxidation of NADH is a key contributor to high oxygen requirements of oxygen-limited cultures of Ogataea parapolymorpha.

While thermotolerance is an attractive trait for yeasts used in industrial ethanol production, oxygen requirements of known thermotolerant species are incompatible with process requirements. Analysis of oxygen-sufficient and oxygen-limited chemostat cultures of the facultatively fermentative, thermotolerant species Ogataea parapolymorpha showed its minimum oxygen requirements to be an order of magnitude larger than those reported for the thermotolerant yeast Kluyveromyces marxianus. High oxygen requirements of O.

Pathway engineering strategies for improved product yield in yeast-based industrial ethanol production.

Product yield on carbohydrate feedstocks is a key performance indicator for industrial ethanol production with the yeast . This paper reviews pathway engineering strategies for improving ethanol yield on glucose and/or sucrose in anaerobic cultures of this yeast by altering the ratio of ethanol production, yeast growth and glycerol formation. Particular attention is paid to strategies aimed at altering energy coupling of alcoholic fermentation and to strategies for altering redox-cofactor coupling in carbon and nitrogen metabolism that aim to reduce or eliminate the role of glycerol formation in anaerobic redox metabolism.

Evolutionary engineering reveals amino acid substitutions in Ato2 and Ato3 that allow improved growth of Saccharomyces cerevisiae on lactic acid.

In Saccharomyces cerevisiae, the complete set of proteins involved in transport of lactic acid across the cell membrane has not been determined. In this study, we aimed to identify transport proteins not previously described to be involved in lactic acid transport via a combination of directed evolution, whole-genome resequencing and reverse engineering. Evolution of a strain lacking all known lactic acid transporters on lactate led to the discovery of mutated Ato2 and Ato3 as two novel lactic acid transport proteins.

Endocytosis of nutrient transporters in fungi: The ART of connecting signaling and trafficking.

Plasma membrane transporters play pivotal roles in the import of nutrients, including sugars, amino acids, nucleobases, carboxylic acids, and metal ions, that surround fungal cells. The selective removal of these transporters by endocytosis is one of the most important regulatory mechanisms that ensures a rapid adaptation of cells to the changing environment (e.g.

Critical Effects on Akt Signaling in Adult Zebrafish Brain Following Alterations in Light Exposure.

Evidence from human and animal studies indicate that disrupted light cycles leads to alterations of the sleep state, poor cognition, and the risk of developing neuroinflammatory and generalized health disorders. Zebrafish exhibit a diurnal circadian rhythm and are an increasingly popular model in studies of neurophysiology and neuropathophysiology. Here, we investigate the effect of alterations in light cycle on the adult zebrafish brain: we measured the effect of altered, unpredictable light exposure in adult zebrafish telencephalon, homologous to mammalian hippocampus, and the optic tectum, a significant visual processing center with extensive telencephalon connections.

Energy coupling of membrane transport and efficiency of sucrose dissimilation in yeast.

Proton coupled transport of α-glucosides via Mal11 into Saccharomyces cerevisiae costs one ATP per imported molecule. Targeted mutation of all three acidic residues in the active site resulted in sugar uniport, but expression of these mutant transporters in yeast did not enable growth on sucrose. We then isolated six unique transporter variants of these mutants by directed evolution of yeast for growth on sucrose.

Regulatory control circuits for stabilizing long-term anabolic product formation in yeast.

Engineering living cells for production of chemicals, enzymes and therapeutics can burden cells due to use of limited native co-factor availability and/or expression burdens, totalling a fitness deficit compared to parental cells encoded through long evolutionary trajectories to maximise fitness. Ultimately, this discrepancy puts a selective pressure against fitness-burdened engineered cells under prolonged bioprocesses, and potentially leads to complete eradication of high-performing engineered cells at the population level. Here we present the mutation landscapes of fitness-burdened yeast cells engineered for vanillin-β-glucoside production.

Engineering for Succinic Acid Production From Glycerol and Carbon Dioxide.

Previously, our lab replaced the endogenous FAD-dependent pathway for glycerol catabolism in by the synthetic NAD-dependent dihydroxyacetone (DHA) pathway. The respective modifications allow the full exploitation of glycerol's higher reducing power (compared to sugars) for the production of the platform chemical succinic acid (SA) via a reductive, carbon dioxide fixing and redox-neutral pathway in a production host robust for organic acid production. Expression cassettes for three enzymes converting oxaloacetate to SA in the cytosol ("SA module") were integrated into the genome of -DHA, an optimized CEN.

Contribution of Complex I NADH Dehydrogenase to Respiratory Energy Coupling in Glucose-Grown Cultures of .

The thermotolerant yeast (formerly ) is an industrially relevant production host that exhibits a fully respiratory sugar metabolism in aerobic batch cultures. NADH-derived electrons can enter its mitochondrial respiratory chain either via a proton-translocating complex I NADH-dehydrogenase or via three putative alternative NADH dehydrogenases. This respiratory entry point affects the amount of ATP produced per NADH/O consumed and therefore impacts the maximum yield of biomass and/or cellular products from a given amount of substrate.

Weak Acid Permeation in Synthetic Lipid Vesicles and Across the Yeast Plasma Membrane.

We present a fluorescence-based approach for determination of the permeability of small molecules across the membranes of lipid vesicles and living cells. With properly designed experiments, the method allows us to assess the membrane physical properties both in vitro and in vivo. We find that the permeability of weak acids increases in the order of benzoic > acetic > formic > lactic, both in synthetic lipid vesicles and the plasma membrane of Saccharomyces cerevisiae, but the permeability is much lower in yeast (one to two orders of magnitude).

Functional expression of a bacterial α-ketoglutarate dehydrogenase in the cytosol of Saccharomyces cerevisiae.

Efficient production of fuels and chemicals by metabolically engineered micro-organisms requires availability of precursor molecules for product pathways. In eukaryotic cell factories, heterologous product pathways are usually expressed in the cytosol, which may limit availability of precursors that are generated in other cellular compartments. In Saccharomyces cerevisiae, synthesis of the precursor molecule succinyl-Coenzyme A is confined to the mitochondrial matrix.

Cholinergic Stimulation of the Adult Zebrafish Brain Induces Phosphorylation of Glycogen Synthase Kinase-3 β and Extracellular Signal-Regulated Kinase in the Telencephalon.

The sequencing of the zebrafish genome, the emergence of powerful gene-editing tools, and the development of imaging techniques have propelled the economical zebrafish into prominence as a biomedical research model. Neurodegenerative disorders with a cholinergic component, such as Alzheimer's and Parkinson's diseases, are currently modeled using zebrafish. Still, the utility of zebrafish as a research model will not be fully realized until their neurophysiological properties are thoroughly characterized.