Accurate and unambiguous tag-to-gene mapping in serial analysis of gene expression

BACKGROUND: In this study, we present a robust and reliable computational method for tag-to-gene assignment in serial analysis of gene expression (SAGE). The method relies on current genome information and annotation, incorporation of several new features, and key improvements over alternative methods, all of which are important to determine gene expression levels more accurately. The method provides a complete annotation of potential virtual SAGE tags within a genome, along with an estimation of their confidence for experimental observation that ranks tags that present multiple matches in the genome. RESULTS: We applied this method to the Saccharomyces cerevisiae genome, producing the most thorough and accurate annotation of potential virtual SAGE tags that is available today for this organism. The usefulness of this method is exemplified by the significant reduction of ambiguous cases in existing experimental SAGE data. In addition, we report new insights from the analysis of existing SAGE data. First, we found that experimental SAGE tags mapping onto introns, intron-exon boundaries, and non-coding RNA elements are observed in all available SAGE data. Second, a significant fraction of experimental SAGE tags was found to map onto genomic regions currently annotated as intergenic. Third, a significant number of existing experimental SAGE tags for yeast has been derived from truncated cDNAs, which are synthesized through oligo-d(T) priming to internal poly-(A) regions during reverse transcription. CONCLUSION: We conclude that an accurate and unambiguous tag mapping process is essential to increase the quality and the amount of information that can be extracted from SAGE experiments. This is supported by the results obtained here and also by the large impact that the erroneous interpretation of these data could have on downstream applications

Sustainable production of β-Xanthophylls in Saccharomyces Cerevisiae

Xanthophylls are a group of C40 pigments that belong to the carotenoids family. β-Xanthophylls, such as zeaxanthin, violaxanthin and neoxanthin are derived from β-carotene metabolism, and play a central role in the protection of photo-oxidative damage in plants and algae. These molecules have interesting applications as precursors of commercially relevant natural aromas, like safranal and damascenone. Furthermore, zeaxanthin is also widely used as a nutraceutical to improve ocular health. In this study, we engineered the yeast Saccharomyces cerevisiae to biosynthesize zeaxanthin and violaxanthin from glucose. We used integrative vectors to construct a genetic stable β-xanthophylls pathway in a β-carotenogenic yeast strain. To find an effective zeaxanthin biosynthetic enzyme, we compared the titers achieved by bacterial, plant and algal β-carotene hydroxylases. Additionally, we evaluated the effect of the chloroplast transit peptide of plant and algal enzymes on zeaxanthin biosynthesis. The strain that expressed truncated version of Solanum lycopersicum β-carotene hydroxylase showed the best performance, reaching up to 4.7 mg/g DCW of zeaxanthin after 72 h cultivation in shake-flasks. Zeaxanthin producing strains were transformed with zeaxanthin epoxidase genes to further extend the pathway to violaxanthin, which was measured by UPLC-MS. To the best of our knowledge, this work presents the highest titer of zeaxanthin in S. cerevisiae reported to date, the first zeaxanthin cell factory using β-carotene hydroxylase from plants, and the first heterologous biosynthesis of violaxanthin. Financial support of FONDECYT grant No.1170745 is greatly acknowledged. Vicente F. Cataldo acknowledges CONICYT for receiving graduate scholarship

Contextualized genome-scale model unveils high-order metabolic effects of the specific growth rate and oxygenation level in recombinant Pichia pastoris

Pichia pastoris is recognized as a biotechnological workhorse for recombinant protein expression. The metabolic performance of this microorganism depends on genetic makeup and culture conditions, amongst which the specific growth rate and oxygenation level are critical. Despite their importance, only their individual effects have been assessed so far, and thus their combined effects and metabolic consequences still remain to be elucidated. In this work, we present a comprehensive framework for revealing high-order (i.e., individual and combined) metabolic effects of the above parameters in glucose-limited continuous cultures of P. pastoris, using thaumatin production as a case study. Specifically, we employed a rational experimental design to calculate statistically significant metabolic effects from multiple chemostat data, which were later contextualized using a refined and highly predictive genome-scale metabolic model of this yeast under the simulated conditions. Our results revealed a negative effect of the oxygenation on the specific product formation rate (thaumatin), and a positive effect on the biomass yield. Notably, we identified a novel positive combined effect of both the specific growth rate and oxygenation level on the specific product formation rate. Finally, model predictions indicated an opposite relationship between the oxygenation level and the growth-associated maintenance energy (GAME) requirement, suggesting a linear GAME decrease of 0.56 mmol ATP/g per each 1% increase in oxygenation level, which translated into a 44% higher metabolic cost under low oxygenation compared to high oxygenation. Overall, this work provides a systematic framework for mapping high-order metabolic effects of different culture parameters on the performance of a microbial cell factory. Particularly in this case, it provided valuable insights about optimal operational conditions for protein production in P. pastoris

Development of an International Odor Identification Test for Children: The Universal Sniff Test

Objective: To assess olfactory function in children and to create and validate an odor identification test to diagnose olfactory dysfunction in children, which we called the Universal Sniff (U-Sniff) test.  Study design: This is a multicenter study involving 19 countries. The U-Sniff test was developed in 3 phases including 1760 children age 5-7 years. Phase 1: identification of potentially recognizable odors; phase 2: selection of odorants for the odor identification test; and phase 3: evaluation of the test and acquisition of normative data. Test—retest reliability was evaluated in a subgroup of children (n = 27), and the test was validated using children with congenital anosmia (n = 14).  Results: Twelve odors were familiar to children and, therefore, included in the U-Sniff test. Children scored a mean ± SD of 9.88 ± 1.80 points out of 12. Normative data was obtained and reported for each country. The U-Sniff test demonstrated a high test—retest reliability (r27 = 0.83, P < .001) and enabled discrimination between normosmia and children with congenital anosmia with a sensitivity of 100% and specificity of 86%.  Conclusions: The U-Sniff is a valid and reliable method of testing olfaction in children and can be used internationally