Expanding the genetic toolbox of Rhodotorula toruloides by identification and validation of six novel promoters induced or repressed under nitrogen starvation.

Background: The non-conventional yeast Rhodotorula toruloides is an emerging host organism in biotechnology by merit of its natural capacity to accumulate high levels of carotenoids and intracellular storage lipids from a variety of carbon sources. While the number of genetic engineering strategies that employ R. toruloides is increasing, the lack of genetic tools available for modification of this yeast is still limiting strain development.

Cinnamic acid and p-coumaric acid are metabolized to 4-hydroxybenzoic acid by Yarrowia lipolytica.

Yarrowia lipolytica has been explored as a potential production host for flavonoid synthesis due to its high tolerance to aromatic acids and ability to supply malonyl-CoA. However, little is known about its ability to consume the precursors cinnamic and p-coumaric acid. In this study, we demonstrate that Y.

Functional genome annotation and transcriptome analysis of Pseudozyma hubeiensis BOT-O, an oleaginous yeast that utilizes glucose and xylose at equal rates.

Pseudozyma hubeiensis is a basidiomycete yeast that has the highly desirable traits for lignocellulose valorisation of being equally efficient at utilization of glucose and xylose, and capable of their co-utilization. The species has previously mainly been studied for its capacity to produce secreted biosurfactants in the form of mannosylerythritol lipids, but it is also an oleaginous species capable of accumulating high levels of triacylglycerol storage lipids during nutrient starvation. In this study, we aimed to further characterize the oleaginous nature of P.

Altering the fatty acid profile of Yarrowia lipolytica to mimic cocoa butter by genetic engineering of desaturases.

Background: Demand for Cocoa butter is steadily increasing, but the supply of cocoa beans is naturally limited and under threat from global warming. One route to meeting the future demand for cocoa butter equivalent (CBE) could be to utilize microbial cell factories such as the oleaginous yeast Yarrowia lipolytica.

Results: The main goal was to achieve triacyl-glycerol (TAG) storage lipids in Y.

Tolerance of Yarrowia lipolytica to inhibitors commonly found in lignocellulosic hydrolysates.

Background: Lignocellulosic material is a suitable renewable carbon and energy source for microbial cell factories, such as Yarrowia lipolytica. To be accessible for microorganisms, the constituent sugars need to be released in a hydrolysis step, which as a side effect leads to the formation of various inhibitory compounds. However, the effects of these inhibitory compounds on the growth of Y.

Deletion of MHY1 abolishes hyphae formation in Yarrowia lipolytica without negative effects on stress tolerance.

There is a need for development of sustainable production processes for production of fats/oils and lipid derived chemicals. The dimorphic oleaginous yeast Yarrowia lipolytica is a promising organism for conversion of biomass hydrolysate to lipids, but in many such processes hyphae formation will be problematic. We have therefore constructed and compared the performance of strains carrying deletions in several published gene targets suggested to abolish hyphae formation (MHY1, HOY1 and CLA4).

A simple kinetic analysis of syngas during steam hydrogasification of biomass using a novel inverted batch reactor with instant high pressure feeding.

A newly designed inverted batch reactor equipped with a pressure-driven feeding system was built for investigating the kinetics of syngas during the steam hydrogasification (SHR) of biomass. The system could instantly load the feedstock into the reactor at high temperature and pressure, which simulated the way to transport the feedstock into a hot and pressurized gasifier. Experiments were conducted from 600°C to 700°C.

Comparative sequence analysis and mutagenesis of ethylene forming enzyme (EFE) 2-oxoglutarate/Fe(II)-dependent dioxygenase homologs.

Background: Ethylene is one of the most used chemical monomers derived from non-renewable sources and we are investigating the possibility of producing it in yeast via the ethylene forming enzyme (EFE) from Pseudomonas syringae. To enable engineering strategies to improve the enzyme, it is necessary to identify the regions and amino acid residues involved in ethylene formation.

Results: We identified the open reading frame for the EFE homolog in Penicillium digitatum and also showed its capability of mediating ethylene production in yeast.

Ethylene production in relation to nitrogen metabolism in Saccharomyces cerevisiae.

We have previously shown that ethylene production in Saccharomyces cerevisiae expressing the ethylene-forming enzyme (EFE) from Pseudomonas syringae is strongly influenced by variations in the mode of cultivation as well as the choice of nitrogen source. Here, we have studied the influence of nitrogen metabolism on the production of ethylene further. Using ammonium, glutamate, glutamate/arginine, and arginine as nitrogen sources, it was found that glutamate (with or without arginine) correlates with a high ethylene production, most likely linked to an observed increase in 2-oxoglutarate levels.

Production of 2-butanol through meso-2,3-butanediol consumption in lactic acid bacteria.

2-Butanol has been an issue of industries in many areas, for example, biofuel production (as an advanced alternate fuel), fermented beverages, and food (as taste-altering component). Thus, its source of production, the biological pathway, and the enzymes involved are of high interest. In this study, 42 different isolates of lactic acid bacteria from nine different species were screened for their capability to consume meso-2,3-butanediol and produce 2-butanol.

2-Butanol and butanone production in Saccharomyces cerevisiae through combination of a B12 dependent dehydratase and a secondary alcohol dehydrogenase using a TEV-based expression system.

2-Butanol and its chemical precursor butanone (methyl ethyl ketone--MEK) are chemicals with potential uses as biofuels and biocommodity chemicals. In order to produce 2-butanol, we have demonstrated the utility of using a TEV-protease based expression system to achieve equimolar expression of the individual subunits of the two protein complexes involved in the B12-dependent dehydratase step (from the pdu-operon of Lactobacillus reuteri), which catalyze the conversion of meso-2,3-butanediol to butanone. We have furthermore identified a NADH dependent secondary alcohol dehydrogenase (Sadh from Gordonia sp.

The influence of health disparities on targeting cancer prevention efforts.

Despite the advances in cancer medicine and the resultant 20% decline in cancer death rates for Americans since 1991, there remain distinct cancer health disparities among African Americans, Hispanics, Native Americans, and the those living in poverty. Minorities and the poor continue to bear the disproportionate burden of cancer, especially in terms of stage at diagnosis, incidence, and mortality. Cancer health disparities are persistent reminders that state-of-the-art cancer prevention, diagnosis, and treatment are not equally effective for and accessible to all Americans.

Identification of factors for improved ethylene production via the ethylene forming enzyme in chemostat cultures of Saccharomyces cerevisiae.

Background: Biotechnological production of the traditional petrochemical ethylene is presently being explored using yeasts as well as bacteria. In this study we quantify the specific ethylene production levels at different conditions in continuous (chemostat) cultivation of Saccharomyces cerevisae expressing the ethylene forming enzyme (EFE) from Pseudomonas syringae.

Results: Our study shows that oxygen availability is an important factor for the ethylene formation.

Physiological adaptations of Saccharomyces cerevisiae evolved for improved butanol tolerance.

Background: Butanol is a chemical with potential uses as biofuel and solvent, which can be produced by microbial fermentation. However, the end product toxicity is one of the main obstacles for developing the production process irrespective of the choice of production organism. The long-term goal of the present project is to produce 2-butanol in Saccharomyces cerevisiae.

Health profiles of adolescents in foster care.

The purpose of this paper is to describe health profiles of adolescents in foster care. The Child Health and Illness Profile-Adolescent Edition clustered adolescents in foster care into 13 mutually exclusive health profiles using dimensions of satisfaction with health, risks, resilience, and discomfort. Health profiles were further characterized into four health status rankings from best to worst health status.

Flux balance analysis for ethylene formation in genetically engineered Saccharomyces cerevisiae.

Biosynthesis of ethylene (ethene) is mainly performed by plants and some bacteria and fungi, via two distinct metabolic routes. Plants use two steps, starting with S-adenosylmethionine, while the ethylene-forming microbes perform an oxygen dependent reaction using 2-oxoglutarate and arginine. Introduction of these systems into Saccharomyces cerevisiae was studied in silico.

Recruitment and retention strategies for minority or poor clinical research participants: lessons from the Healthy Aging in Neighborhoods of Diversity across the Life Span study.

Purpose Of The Study: Investigating health disparities requires studies designed to recruit and retain racially and socioeconomically diverse cohorts. It is critical to address the barriers that disproportionately affect participation in clinical research by minorities and the socioeconomically disadvantaged. This study sought to identify and rectify these barriers to recruit and retain a biracial (African American and non-Hispanic White) and socioeconomically diverse cohort for a longitudinal study.

Dimensions of health in young people in foster care.

Unlabelled: To describe the dimensions of health and illness from the perspective of adolescents in foster care.

Methods: Descriptive analyses of dimensions of health were conducted on N = 105 adolescents in foster care. Differences among demographic (age, gender, race/ethnicity) and foster care placement (age at first placement, reason(s) for foster care placement, length of time in care, number, and types of placement) variables and the dimensions and subdimensions of health (Child Health and Illness Profile- Adolescent Edition) were determined using T-tests and ANOVA.

A QPCR-based reporter system to study post-transcriptional regulation via the 3' untranslated region of mRNA in Saccharomyces cerevisiae.

Post-transcriptional regulation via the 3' untranslated region (3' UTR) of mRNA is an important factor in governing eukaryotic gene expression. Achieving detailed understanding of these processes requires highly quantitative systems in which comparative studies can be performed. To this end, we have developed a plasmid reporter system for Saccharomyces cerevisiae, in which the 3' UTR can be easily replaced and modified.

Carbon source dependent dynamics of the Ccr4-Not complex in Saccharomyces cerevisiae.

We have investigated the composition of the conserved Ccr4-Not complex during different physiological states of Saccharomyces cerevisiae. Major changes were found, most notably in the expression of the central scaffold protein Not1p, which was strongly reduced in the absence of glucose. The low expression of Not1p was also evident from the inability of Pop2p to co-purify Not1p in cells from cultures lacking glucose.

Ethylene production by metabolic engineering of the yeast Saccharomyces cerevisiae.

The non-ethylene producing yeast, Saccharomyces cerevisiae, was transformed into an ethylene producer by introducing the ethylene forming enzyme from the plant pathogenic bacterium Pseudomonas syringae. Cultivation of the metabolically engineered strain was performed in well-controlled bioreactors as aerobic batch cultures with an on-line monitoring of ethylene production. The highest productivity was obtained during the respiro-fermentative growth on glucose but there was also a significant rate of formation during the subsequent phase of ethanol respiration.

Comparison of the proteomes of three yeast wild type strains: CEN.PK2, FY1679 and W303.

Yeast deletion strains created during gene function analysis projects very often show drastic phenotypic differences depending on the genetic background used. These results indicate the existence of important molecular differences between the CEN.PK2, FY1679 and W303 wild type strains.

A complete inventory of all enzymes in the eukaryotic methionine salvage pathway.

The methionine salvage pathway is universally used to regenerate methionine from 5'-methylthioadenosine, a byproduct of certain reactions involving S-adenosylmethionine. We identified and verified the genes encoding the enzymes of all steps in this cycle in a commonly used eukaryotic model system: the yeast Saccharomyces cerevisiae. The genes encoding 5'-methylthioribose-1-phosphate isomerase and 5'-methylthioribulose-1-phosphate dehydratase are herein named MRI1 and MDE1, respectively.

Immuno-qPCR detection of the tandem affinity purification (TAP)-tag as a sensitive and accurate tool suitable for large-scale protein quantification.

The tandem affinity purification (TAP)-tag has rapidly gained a wide popularity, mostly in studies on protein interactions, but lately also in large-scale protein quantification studies. We have developed an immuno-quantitative real-time PCR (qPCR) method to achieve rapid, sensitive and accurate quantification of TAP-tagged (and protein A-tagged) proteins in yeast with a detection range between 10(7) and 10(10) molecules. The immuno-qPCR protein quantification showed an excellent correlation to the published in vivo fluorescent protein (GFP)-based large-scale protein quantifications, but allowed for a much higher sensitivity.

Measurements of in-use emissions from modern vehicles using an on-board measurement system.

Emissions from "low emitting" modern vehicles were measured on-road using a Fourier transform infrared (FTIR) on-board emissions measurement system. Twenty vehicles were tested on road and on a chassis dynamometer. A subset of four vehicles was tested on a test track as well as on the dynamometer.