Identification of novel genes in the carotenogenic and oleaginous yeast Rhodotorula toruloides through genome-wide insertional mutagenesis.

BACKGROUND: Rhodotorula toruloides is an outstanding producer of lipids and carotenoids. Currently, information on the key metabolic pathways and their molecular basis of regulation remains scarce, severely limiting efforts to engineer it as an industrial host. RESULTS: We have adapted Agrobacterium tumefaciens-mediated transformation (ATMT) as a gene-tagging tool for the identification of novel genes in R. toruloides. Multiple factors affecting transformation efficiency in several species in the Pucciniomycotina subphylum were optimized. The Agrobacterium transfer DNA (T-DNA) showed predominantly single-copy chromosomal integrations in R. toruloides, which were trackable by high efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR). To demonstrate the application of random T-DNA insertions for strain improvement and gene hunting, 3 T-DNA insertional libraries were screened against cerulenin, nile red and tetrazolium violet respectively, resulting in the identification of 22 mutants with obvious phenotypes in fatty acid or lipid metabolism. Similarly, 5 carotenoid biosynthetic mutants were obtained through visual screening of the transformants. To further validate the gene tagging strategy, one of the carotenoid production mutants, RAM5, was analyzed in detail. The mutant had a T-DNA inserted at the putative phytoene desaturase gene CAR1. Deletion of CAR1 by homologous recombination led to a phenotype similar to RAM5 and it could be genetically complemented by re-introduction of the wild-type CAR1 genome sequence. CONCLUSIONS: T-DNA insertional mutagenesis is an efficient forward genetic tool for gene discovery in R. toruloides and related oleaginous yeast species. It is also valuable for metabolic engineering in these hosts. Further analysis of the 27 mutants identified in this study should augment our knowledge of the lipid and carotenoid biosynthesis, which may be exploited for oil and isoprenoid metabolic engineering.

Molecular insights into 4-nitrophenol-induced hepatotoxicity in zebrafish: transcriptomic, histological and targeted gene expression analyses.

BACKGROUND: 4-Nitrophenol (4-NP) is a prioritized environmental pollutant and its toxicity has been investigated using zebrafish, advocated as an alternative toxicological model. However, molecular information of 4-NP induced hepatotoxicity is still limited. This study aimed to obtain molecular insights into 4-NP-induced hepatotoxicity using zebrafish as a model. METHODS: Adult male zebrafish were exposed to 4-NP for 8, 24, 48 and 96h. Livers were sampled for microarray experiment, qRT-PCR and various histological analyses. RESULTS: Transcriptomic analysis revealed that genes associated with oxidative phosphorylation and electron transport chain were significantly up-regulated throughout early and late stages of 4-NP exposure due to oxidative phosphorylation uncoupling by 4-NP. This in turn induced oxidative stress damage and up-regulated pathways associated with tumor suppressors Rb and p53, cell cycle, DNA damage, proteasome degradation and apoptosis. Pathways associated with cell adhesion and morphology were deregulated. Carbohydrate and lipid metabolisms were down-regulated while methionine and aromatic amino acid metabolisms as well as NFKB pathway associated with chronic liver conditions were up-regulated. Up-regulation of NFKB, NFAT and interleukin pathways suggested hepatitis. Histological analyses with specific staining methods and qRT-PCR analysis of selected genes corroborated with the transcriptomic analysis suggesting 4-NP induced liver injury. CONCLUSION: Our findings allowed us to propose a plausible model and provide a broader understanding of the molecular events leading to 4-NP induced acute hepatotoxicity for future studies involving other nitrophenol derivatives. GENERAL SIGNIFICANCE: This is the first transcriptomic report on 4-NP induced hepatotoxicity.

Characterization of glyceraldehyde-3-phosphate dehydrogenase gene RtGPD1 and development of genetic transformation method by dominant selection in oleaginous yeast Rhodosporidium toruloides.

The oleaginous yeast Rhodosporidium toruloides, which belongs to the Pucciniomycotina subphylum in the Basidiomycota, has attracted strong interest in the biofuel community recently due to its ability to accumulate more than 60% of dry biomass as lipid under high-density fermentation. A 3,543-nucleotide (nt) DNA fragment of the glyceraldehyde-3-phosphate dehydrogenase gene (GPD1) was isolated from R. toruloides ATCC 10657 and characterized in details. The 1,038-nt mRNA derived from seven exons encodes an open reading frame (ORF) of 345 amino acids that shows high identity (80%) to the Ustilago maydis homolog. Notably, the ORF is composed of codons strongly biased towards cytosine at the Wobble position. GPD1 is transcriptionally regulated by temperature shock, osmotic stress, and carbon source. Nested deletion analysis of the GPD1 promoter by GFP reporter assay revealed that two regions, -975 to -1,270 and -1,270 to -1,429, upstream from the translational start site of GPD1 were important for responses to various stress stimuli. Interestingly, a 176-bp short fragment maintained 42.2% promoter activity of the 795-bp version in U. maydis whereas it was reduced to 17.4% in R. toruloides. The GPD1 promoter drove strong expression of a codon-optimized enhanced green fluorescent protein gene (RtGFP) and a codon-optimized hygromycin phosphotransferase gene (hpt-3), which was critical for Agrobacterium tumefaciens-mediated transformation in R. toruloides.

Mercury-induced hepatotoxicity in zebrafish: in vivo mechanistic insights from transcriptome analysis, phenotype anchoring and targeted gene expression validation.

BACKGROUND: Mercury is a prominent environmental contaminant that causes detrimental effects to human health. Although the liver has been known to be a main target organ, there is limited information on in vivo molecular mechanism of mercury-induced toxicity in the liver. By using transcriptome analysis, phenotypic anchoring and validation of targeted gene expression in zebrafish, mercury-induced hepatotoxicity was investigated and a number of perturbed cellular processes were identified and compared with those captured in the in vitro human cell line studies. RESULTS: Hepato-transcriptome analysis of mercury-exposed zebrafish revealed that the earliest deregulated genes were associated with electron transport chain, mitochondrial fatty acid beta-oxidation, nuclear receptor signaling and apoptotic pathway, followed by complement system and proteasome pathway, and thereafter DNA damage, hypoxia, Wnt signaling, fatty acid synthesis, gluconeogenesis, cell cycle and motility. Comparative meta-analysis of microarray data between zebrafish liver and human HepG2 cells exposed to mercury identified some common toxicological effects of mercury-induced hepatotoxicity in both models. Histological analyses of liver from mercury-exposed fish revealed morphological changes of liver parenchyma, decreased nucleated cell count, increased lipid vesicles, glycogen and apoptotic bodies, thus providing phenotypic evidence for anchoring of the transcriptome analysis. Validation of targeted gene expression confirmed deregulated gene-pathways from enrichment analysis. Some of these genes responding to low concentrations of mercury may serve as toxicogenomic-based markers for detection and health risk assessment of environmental mercury contaminations. CONCLUSION: Mercury-induced hepatotoxicity was triggered by oxidative stresses, intrinsic apoptotic pathway, deregulation of nuclear receptor and kinase activities including Gsk3 that deregulates Wnt signaling pathway, gluconeogenesis, and adipogenesis, leading to mitochondrial dysfunction, endocrine disruption and metabolic disorders. This study provides important mechanistic insights into mercury-induced liver toxicity in a whole-animal physiology context, which will help in understanding the syndromes caused by mercury poisoning. The molecular conservation of mercury-induced hepatotoxicity between zebrafish and human cell line reveals the feasibility of using zebrafish to model molecular toxicity in human for toxicant risk assessments.

Oxidative stability of spray dried matcha-tuna oil powders.

Matcha-tuna oil and matcha-maltodextrin-tuna oil emulsions (25% oil, dry basis), formulated to have protein: carbohydrate ratios of 1:1.1, 1:2, 1:3 and 1:4, were spray dried. Confocal laser scanning microscopy showed effective emulsification of oil in all emulsions. All powders had low surface fat (2.9-4.2%). The addition of maltodextrin enhanced the bulk density and flowability of powders. Water sorption isotherms indicated that addition of maltodextrin increased water uptake of powders. The oxidative stability of the powders under accelerated conditions in an Oxipres® was highest for the matcha-tuna oil powder. Increasing amounts of added maltodextrin decreased oxidative stability. A comparison of levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in neat oil and tuna oil powders over 12 weeks at 40 °C, demonstrated that % remaining EPA and DHA were higher for all spray dried powders compared to neat oil. There was a significant correlation (p < 0.01) between the amount of the loss of tea catechins and % remaining EPA and DHA after 12 weeks at 40 °C, suggesting that the catechins had a major role in protecting the tuna oil against oxidation. This study has demonstrated the potential of using a whole biomass (matcha) as the single encapsulant for protection and delivery of omega-3 oils.

Fermentation by Probiotic Lactobacillus gasseri Strains Enhances the Carotenoid and Fibre Contents of Carrot Juice.

Carrot juice (straight, 8.5 Brix and concentrated, 15.2 Brix) was fermented by lactic acid bacteria (Lactobacillus gasseri strain DSM 20604 or DSM 20077). Fermentation enhanced the nutritional profile of carrot juice. There was a greater sugar reduction (27%) in fermented straight carrot juices than in the fermented concentrated juices (15%). The sugar reduction was independent of the strain used for fermentation. The two L. gasseri strains synthesised fructosyltransferase enzymes during fermentation of carrot juice samples that enabled conversion of simple sugars primarily into polysaccharides. The level of conversion to polysaccharides was dependent on the L. gasseri strain and juice concentration. Fermentation of carrot juice by L. gasseri enables the production of a nutritionally-enhanced beverage with reduced calorie and prebiotic potential. An additional benefit is the increased carotenoid content observed in straight and concentrated juices fermented by Lactobacillus gasseri DSM 20077 and the concentrated juice fermented by Lactobacillus gasseri DSM 20604.

Development of broccoli by-products as carriers for delivering EGCG.

Novel delivery systems for epigallocatechin gallate (EGCG) were developed using broccoli by-products and their fractions as carriers. Puree and pomace from broccoli by-products had higher adsorption capacities for EGCG than juice at 25 °C (43.20 mg g-1, 39.47 mg g-1 and 25.22 mg g-1 dry weight for pomace, puree and juice respectively). Chemical sorption is the rate-controlling step for EGCG-broccoli interactions. Langmuir and Freundlich models well described the adsorption of EGCG onto puree and pomace. FTIR results indicated that EGCG-puree had stronger interaction than EGCG-pomace. When the same level of EGCG (∼26 mg) was added to different matrices, more EGCG (∼20%) was recovered from the in vitro digestion system of EGCG-loaded puree than from the EGCG-loaded pomace (14%) and neat EGCG (9%). The antioxidant capacity of the whole digesta was positively correlated with the EGCG levels. Broccoli by-products are promising carriers for delivering and stabilizing EGCG through gastrointestinal digestion.

Mammalian specific mouse genes are evolving faster than mouse genes conserved across other eukaryotic lineages.

Positive selection is usually considered in the context of a higher rate of substitutions in non-synonymous as compared to synonymous sites in complete coding sequences of genes or individual positions. We show that genes conserved in eukaryota, coelomata, and bilateria, that is, proteins that arose earlier in evolution as compared to mammalia specific genes evolve slowly and are subjected to negative selection. This finding supports the notion that evolutionary rates progressively diminish with the age of a gene. The data suggests that in both intron-containing and intronless genes synonymous sites may be subject to some degree of selection that is indicative of a relative acceleration of amino-acid substitution, which could be due to a relaxation of functional constraints and/or directional selection.

Physical properties and FTIR analysis of rice-oat flour and maize-oat flour based extruded food products containing olive pomace.

Olive pomace, a waste stream from olive oil processing, was fractionated by centrifugation to obtain a supernatant and a flesh-enriched fraction, and freeze dried to obtain a powder. The dried supernatant contained 5.8% moisture, 4.8% protein, 3.5% fat, 3.5% ash, 82.4% carbohydrate (including 17.2% dietary fiber) and polyphenols (2970mg gallic acid equivalents (GAE)/100g). The dried flesh-enriched fraction, contained 5.9% moisture, 13.4% protein, 14.2% fat, 3.5% ash, 63.1% carbohydrate (including 42.7% dietary fiber) and polyphenols (1960mg GAE/100g). The extruded products using rice-oat flour or maize-oat flour mixtures as the base were formulated to contain 5% or 10% olive pomace fractions (dry basis). The extruded products with added olive pomace fractions has higher fiber (2-7g/100g) and polyphenol contents (67-161mg GAE/100g) compared to the corresponding mixtures of rice-oat flour base (0.92g/100g fiber, 20mg GAE/100g) or maize-oat flour base (3.2g/100g fiber, 20mg GAE/100g) without olive pomace fractions. Addition of olive pomace fractions reduced the die pressure and specific mechanical energy during extrusion and resulted in lower radial expansion in the extruded product. The impact of the addition of olive pomace fraction on physical characteristics of the extruded product is higher for rice-oat flour base than maize-oat flour base. The underlining mechanism was explained by FTIR analysis. FTIR showed that there were significant changes in the carbohydrate components and the structure of the proteins on extrusion, with consequent effects on the expansion and density of the extruded product. This study showed the feasibility of preparing fiber and polyphenol enriched extruded products by incorporation of olive pomace. This shows the potential of recovery and diversion of edible components from waste streams of olive oil processing for formulation of extruded products.

Role of Burkholderia pseudomallei Sigma N2 in Amino Acids Utilization and in Regulation of Catalase E Expression at the Transcriptional Level.

Burkholderia pseudomallei is the causative agent of melioidosis. The complete genome sequences of this pathogen have been revealed, which explain some pathogenic mechanisms. In various hostile conditions, for example, during nitrogen and amino acid starvation, bacteria can utilize alternative sigma factors such as RpoS and RpoN to modulate genes expression for their adaptation and survival. In this study, we demonstrate that mutagenesis of rpoN2, which lies on chromosome 2 of B. pseudomallei and encodes a homologue of the sigma factor RpoN, did not alter nitrogen and amino acid utilization of the bacterium. However, introduction of B. pseudomallei rpoN2 into E. coli strain deficient for rpoN restored the ability to utilize amino acids. Moreover, comparative partial proteomic analysis of the B. pseudomallei wild type and its rpoN2 isogenic mutant was performed to elucidate its amino acids utilization property which was comparable to its function found in the complementation assay. By contrast, the rpoN2 mutant exhibited decreased katE expression at the transcriptional and translational levels. Our finding indicates that B. pseudomallei RpoN2 is involved in a specific function in the regulation of catalase E expression.

Toxicogenomic and phenotypic analyses of bisphenol-A early-life exposure toxicity in zebrafish.

Bisphenol-A is an important environmental contaminant due to the increased early-life exposure that may pose significant health-risks to various organisms including humans. This study aimed to use zebrafish as a toxicogenomic model to capture transcriptomic and phenotypic changes for inference of signaling pathways, biological processes, physiological systems and identify potential biomarker genes that are affected by early-life exposure to bisphenol-A. Phenotypic analysis using wild-type zebrafish larvae revealed BPA early-life exposure toxicity caused cardiac edema, cranio-facial abnormality, failure of swimbladder inflation and poor tactile response. Fluorescent imaging analysis using three transgenic lines revealed suppressed neuron branching from the spinal cord, abnormal development of neuromast cells, and suppressed vascularization in the abdominal region. Using knowledge-based data mining algorithms, transcriptome analysis suggests that several signaling pathways involving ephrin receptor, clathrin-mediated endocytosis, synaptic long-term potentiation, axonal guidance, vascular endothelial growth factor, integrin and tight junction were deregulated. Physiological systems with related disorders associated with the nervous, cardiovascular, skeletal-muscular, blood and reproductive systems were implicated, hence corroborated with the phenotypic analysis. Further analysis identified a common set of BPA-targeted genes and revealed a plausible mechanism involving disruption of endocrine-regulated genes and processes in known susceptible tissue-organs. The expression of 28 genes were validated in a separate experiment using quantitative real-time PCR and 6 genes, ncl1, apoeb, mdm1, mycl1b, sp4, U1SNRNPBP homolog, were found to be sensitive and robust biomarkers for BPA early-life exposure toxicity. The susceptibility of sp4 to BPA perturbation suggests its role in altering brain development, function and subsequently behavior observed in laboratory animals exposed to BPA during early life, which is a health-risk concern of early life exposure in humans. The present study further established zebrafish as a model for toxicogenomic inference of early-life chemical exposure toxicity.