A label-free real-time method for measuring glucose uptake kinetics in yeast.

Glucose uptake assays commonly rely on the isotope-labeled sugar, which is associated with radioactive waste and exposure of the experimenter to radiation. Here, we show that the rapid decrease of the cytosolic pH after a glucose pulse to starved Saccharomyces cerevisiae cells is dependent on the rate of sugar uptake and can be used to determine the kinetic parameters of sugar transporters. The pH-sensitive green fluorescent protein variant pHluorin is employed as a genetically encoded biosensor to measure the rate of acidification as a proxy of transport velocity in real time.

Artificial ER-Derived Vesicles as Synthetic Organelles for Compartmentalization of Biochemical Pathways.

Compartmentalization in membrane-surrounded organelles has the potential to overcome obstacles associated with the engineering of metabolic pathways, such as unwanted side reactions, accumulation of toxic intermediates, drain of intermediates out of the cell, and long diffusion distances. Strategies utilizing natural organelles suffer from the presence of endogenous pathways. In our approach, we make use of endoplasmic reticulum-derived vesicles loaded with enzymes of a metabolic pathway ("metabolic vesicles").

A superfolder variant of pH-sensitive pHluorin for in vivo pH measurements in the endoplasmic reticulum.

Many cellular processes are regulated via pH, and maintaining the pH of different organelles is crucial for cell survival. A pH-sensitive GFP variant, the so-called pHluorin, has proven to be a valuable tool to study the pH of the cytosol, mitochondria and other organelles in vivo. We found that the fluorescence intensity of Endoplasmic Reticulum (ER)-targeted pHluorin in the yeast Saccharomyces cerevisiae was very low and barely showed pH sensitivity, probably due to misfolding in the oxidative environment of the ER.

Engineering of hydroxymandelate synthases and the aromatic amino acid pathway enables de novo biosynthesis of mandelic and 4-hydroxymandelic acid with Saccharomyces cerevisiae.

Mandelic acid (MA) and 4-hydroxymandelic acid (HMA) are valuable specialty chemicals used as precursors for flavors as well as for cosmetic and pharmaceutical purposes. Today they are mainly synthesized chemically. Their synthesis through microbial fermentation would allow for environmentally sustainable production.

Pathway engineering for the production of heterologous aromatic chemicals and their derivatives in Saccharomyces cerevisiae: bioconversion from glucose.

Saccharomyces cerevisiae has been extensively engineered for optimising its performance as a microbial cell factory to produce valuable aromatic compounds and their derivatives as bulk and fine chemicals. The production of heterologous aromatic molecules in yeast is achieved via engineering of the aromatic amino acid biosynthetic pathway. This pathway is connected to two pathways of the central carbon metabolism, and is highly regulated at the gene and protein level.

Parallelised online biomass monitoring in shake flasks enables efficient strain and carbon source dependent growth characterisation of Saccharomyces cerevisiae.

Background: Baker's yeast, Saccharomyces cerevisiae, as one of the most often used workhorses in biotechnology has been developed into a huge family of application optimised strains in the last decades. Increasing numbers of strains render their characterisation highly challenging, even with the simple methods of growth-based analytics. Here we present a new sensor system for the automated, non-invasive and parallelisable monitoring of biomass in continuously shaken shake flask cultures, called CGQ ("cell growth quantifier").

Dissociation of a BRICHOS trimer into monomers leads to increased inhibitory effect on Aβ42 fibril formation.

The BRICHOS domain is associated with human amyloid disease, and it efficiently prevents amyloid fibril formation of the amyloid β-peptide (Aβ) in vitro and in vivo. Recombinant human prosurfactant protein C (proSP-C) BRICHOS domain forms a homotrimer as observed by x-ray crystallography, analytical ultracentrifugation, native polyacrylamide gel electrophoresis, analytical size exclusion chromatography and electrospray mass spectrometry. It was hypothesized that the trimer is an inactive storage form, as a putative substrate-binding site identified in the monomer, is buried in the subunit interface of the trimer.