A novel ERß high throughput microscopy platform for testing endocrine disrupting chemicals.

In this study we present an inducible biosensor model for the Estrogen Receptor Beta (ERß), GFP-ERß:PRL-HeLa, a single-cell-based high throughput (HT) in vitro assay that allows direct visualization and measurement of GFP-tagged ERß binding to ER-specific DNA response elements (EREs), ERß-induced chromatin remodeling, and monitor transcriptional alterations via mRNA fluorescence in situ hybridization for a prolactin (PRL)-dsRED2 reporter gene. The model was used to accurately (Z' = 0.58-0.8) differentiate ERß-selective ligands from ERα ligands when treated with a panel of selective agonists and antagonists. Next, we tested an Environmental Protection Agency (EPA)-provided set of 45 estrogenic reference chemicals with known ERα in vivo activity and identified several that activated ERß as well, with varying sensitivity, including a subset that is completely novel. We then used an orthogonal ERE-containing transgenic zebrafish (ZF) model to cross validate ERß and ERα selective activities at the organism level. Using this environmentally relevant ZF assay, some compounds were confirmed to have ERß activity, validating the GFP-ERß:PRL-HeLa assay as a screening tool for potential ERß active endocrine disruptors (EDCs). These data demonstrate the value of sensitive multiplex mechanistic data gathered by the GFP-ERß:PRL-HeLa assay coupled with an orthogonal zebrafish model to rapidly identify environmentally relevant ERß EDCs and improve upon currently available screening tools for this understudied nuclear receptor.

Faster, reduced cost calibration method development methods for the analysis of fermentation product using near-infrared spectroscopy (NIRS).

Recent innovations in synthetic biology, fermentation, and process development have decreased time to market by reducing strain construction cycle time and effort. Faster analytical methods are required to keep pace with these innovations, but current methods of measuring fermentation titers often involve manual intervention and are slow, time-consuming, and difficult to scale. Spectroscopic methods like near-infrared (NIR) spectroscopy address this shortcoming; however, NIR methods require calibration model development that is often costly and time-consuming. Here, we introduce two approaches that speed up calibration model development. First, generalized calibration modeling (GCM) or sibling modeling, which reduces calibration modeling time and cost by up to 50% by reducing the number of samples required. Instead of constructing analyte-specific models, GCM combines a reduced number of spectra from several individual analytes to produce a large pool of spectra for a generalized model predicting all analyte levels. Second, randomized multicomponent multivariate modeling (RMMM) reduces modeling time by mixing multiple analytes into one sample matrix and then taking the spectral measurements. Afterward, individual calibration methods are developed for the various components in the mixture. Time saved from the use of RMMM is proportional to the number of components or analytes in the mixture. When combined, the two methods effectively reduce the associated cost and time for calibration model development by a factor of 10.

Use of pantothenate as a metabolic switch increases the genetic stability of farnesene producing Saccharomyces cerevisiae.

We observed that removing pantothenate (vitamin B5), a precursor to co-enzyme A, from the growth medium of Saccharomyces cerevisiae engineered to produce ß-farnesene reduced the strain׳s farnesene flux by 70%, but increased its viability, growth rate and biomass yield. Conversely, the growth rate and biomass yield of wild-type yeast were reduced. Cultivation in media lacking pantothenate eliminates the growth advantage of low-producing mutants, leading to improved production upon scale-up to lab-scale bioreactor testing. An omics investigation revealed that when exogenous pantothenate levels are limited, acyl-CoA metabolites decrease, ß-oxidation decreases from unexpectedly high levels in the farnesene producer, and sterol and fatty acid synthesis likely limits the growth rate of the wild-type strain. Thus pantothenate supplementation can be utilized as a "metabolic switch" for tuning the synthesis rates of molecules relying on CoA intermediates and aid the economic scale-up of strains producing acyl-CoA derived molecules to manufacturing facilities.

Metabolic engineering of Saccharomyces cerevisiae for production of carboxylic acids: current status and challenges.

To meet the demands of future generations for chemicals and energy and to reduce the environmental footprint of the chemical industry, alternatives for petrochemistry are required. Microbial conversion of renewable feedstocks has a huge potential for cleaner, sustainable industrial production of fuels and chemicals. Microbial production of organic acids is a promising approach for production of chemical building blocks that can replace their petrochemically derived equivalents. Although Saccharomyces cerevisiae does not naturally produce organic acids in large quantities, its robustness, pH tolerance, simple nutrient requirements and long history as an industrial workhorse make it an excellent candidate biocatalyst for such processes. Genetic engineering, along with evolution and selection, has been successfully used to divert carbon from ethanol, the natural endproduct of S. cerevisiae, to pyruvate. Further engineering, which included expression of heterologous enzymes and transporters, yielded strains capable of producing lactate and malate from pyruvate. Besides these metabolic engineering strategies, this review discusses the impact of transport and energetics as well as the tolerance towards these organic acids. In addition to recent progress in engineering S. cerevisiae for organic acid production, the key limitations and challenges are discussed in the context of sustainable industrial production of organic acids from renewable feedstocks.

Anaerobic homolactate fermentation with Saccharomyces cerevisiae results in depletion of ATP and impaired metabolic activity.

Conversion of glucose to lactic acid is stoichiometrically equivalent to ethanol formation with respect to ATP formation from substrate-level phosphorylation, redox equivalents and product yield. However, anaerobic growth cannot be sustained in homolactate fermenting Saccharomyces cerevisiae. ATP-dependent export of the lactate anion and/or proton, resulting in net zero ATP formation, is suspected as the underlying cause. In an effort to understand the mechanisms behind the decreased lactic acid production rate in anaerobic homolactate cultures of S. cerevisiae, aerobic carbon-limited chemostats were performed and subjected to anaerobic perturbations in the presence of high glucose concentrations. Intracellular measurements of adenosine phosphates confirmed ATP depletion and decreased energy charge immediately upon anaerobicity. Unexpectedly, readily available sources of carbon and energy, trehalose and glycogen, were not activated in homolactate strains as they were in reference strains that produce ethanol. Finally, the anticipated increase in maximal velocity (V(max)) of glycolytic enzymes was not observed in homolactate fermentation suggesting the absence of protein synthesis that may be attributed to decreased energy availability. Essentially, anaerobic homolactate fermentation results in energy depletion, which, in turn, hinders protein synthesis, central carbon metabolism and subsequent energy generation.

Catalase overexpression reduces lactic acid-induced oxidative stress in Saccharomyces cerevisiae.

Industrial production of lactic acid with the current pyruvate decarboxylase-negative Saccharomyces cerevisiae strains requires aeration to allow for respiratory generation of ATP to facilitate growth and, even under nongrowing conditions, cellular maintenance. In the current study, we observed an inhibition of aerobic growth in the presence of lactic acid. Unexpectedly, the cyb2Delta reference strain, used to avoid aerobic consumption of lactic acid, had a specific growth rate of 0.25 h(-1) in anaerobic batch cultures containing lactic acid but only 0.16 h(-1) in identical aerobic cultures. Measurements of aerobic cultures of S. cerevisiae showed that the addition of lactic acid to the growth medium resulted in elevated levels of reactive oxygen species (ROS). To reduce the accumulation of lactic acid-induced ROS, cytosolic catalase (CTT1) was overexpressed by replacing the native promoter with the strong constitutive TPI1 promoter. Increased activity of catalase was confirmed and later correlated with decreased levels of ROS and increased specific growth rates in the presence of high lactic acid concentrations. The increased fitness of this genetically modified strain demonstrates the successful attenuation of additional stress that is derived from aerobic metabolism and may provide the basis for enhanced (micro)aerobic production of organic acids in S. cerevisiae.

Physiological and transcriptional responses to high concentrations of lactic acid in anaerobic chemostat cultures of Saccharomyces cerevisiae.

Based on the high acid tolerance and the simple nutritional requirements of Saccharomyces cerevisiae, engineered strains of this yeast are considered biocatalysts for industrial production of high-purity undissociated lactic acid. However, high concentrations of lactic acid are toxic to S. cerevisiae, thus limiting its growth and product formation. Physiological and transcriptional responses to high concentrations of lactic acid were studied in anaerobic, glucose-limited chemostat cultures grown at different pH values and lactic acid concentrations, resulting in a 50% decrease in the biomass yield. At pH 5, the yield decrease was caused mostly by osmotically induced glycerol production and not by the classic weak-acid action, as was observed at pH 3. Cultures grown at pH 5 with 900 mM lactic acid revealed an upregulation of many genes involved in iron homeostasis, indicating that iron chelation occurred at high concentrations of dissociated lactic acid. Chemostat cultivation at pH 3 with 500 mM lactate, resulting in lower anion concentrations, showed an alleviation of this iron homeostasis response. Six of the 10 known targets of the transcriptional regulator Haa1p were strongly upregulated in lactate-challenged cultures at pH 3 but showed only moderate induction by high lactate concentrations at pH 5. Moreover, the haa1Delta mutant exhibited a growth defect at high lactic acid concentrations at pH 3. These results indicate that iron homeostasis plays a major role in the response of S. cerevisiae to high lactate concentrations, whereas the Haa1p regulon is involved primarily in the response to high concentrations of undissociated lactic acid.

Generic and specific transcriptional responses to different weak organic acids in anaerobic chemostat cultures of Saccharomyces cerevisiae.

Transcriptional responses to four weak organic acids (benzoate, sorbate, acetate and propionate) were investigated in anaerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae. To enable quantitative comparison of the responses to the acids, their concentrations were chosen such that they caused a 50% decrease of the biomass yield on glucose. The concentration of each acid required to achieve this yield was negatively correlated with membrane affinity. Microarray analysis revealed that each acid caused hundreds of transcripts to change by over twofold relative to reference cultures without added organic acids. However, only 14 genes were consistently upregulated in response to all acids. The moderately lipophilic compounds benzoate and sorbate and, to a lesser extent, the less lipophilic acids acetate and propionate showed overlapping transcriptional responses. Statistical analysis for overrepresented functional categories and upstream regulatory elements indicated that responses to the strongly lipophilic acids were focused on genes related to the cell wall, while acetate and propionate had a stronger impact on membrane-associated transport processes. The fact that S. cerevisiae exhibits a minimal generic transcriptional response to weak organic acids along with extensive specific responses is relevant for interpreting and controlling weak acid toxicity in food products and in industrial fermentation processes.

Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status.

Fuel ethanol production from plant biomass hydrolysates by Saccharomyces cerevisiae is of great economic and environmental significance. This paper reviews the current status with respect to alcoholic fermentation of the main plant biomass-derived monosaccharides by this yeast. Wild-type S. cerevisiae strains readily ferment glucose, mannose and fructose via the Embden-Meyerhof pathway of glycolysis, while galactose is fermented via the Leloir pathway. Construction of yeast strains that efficiently convert other potentially fermentable substrates in plant biomass hydrolysates into ethanol is a major challenge in metabolic engineering. The most abundant of these compounds is xylose. Recent metabolic and evolutionary engineering studies on S. cerevisiae strains that express a fungal xylose isomerase have enabled the rapid and efficient anaerobic fermentation of this pentose. L: -Arabinose fermentation, based on the expression of a prokaryotic pathway in S. cerevisiae, has also been established, but needs further optimization before it can be considered for industrial implementation. In addition to these already investigated strategies, possible approaches for metabolic engineering of galacturonic acid and rhamnose fermentation by S. cerevisiae are discussed. An emerging and major challenge is to achieve the rapid transition from proof-of-principle experiments under 'academic' conditions (synthetic media, single substrates or simple substrate mixtures, absence of toxic inhibitors) towards efficient conversion of complex industrial substrate mixtures that contain synergistically acting inhibitors.

Buffering capacity of whole corn mash alters concentrations of organic acids required to inhibit growth of Saccharomyces cerevisiae and ethanol production.

Growth of Saccharomyces cerevisiae and fermentative ethanol production in the presence of acetic and lactic acids was measured in whole corn mash. In this industrial medium, as compared to glucose minimal medium, the yeast had increased tolerance to organic acid stress. It was concluded that the increased buffering capacity of whole corn mash, resulting in decreased concentration of undissociated acid, was responsible for this phenomenon.