Developing a Yeast Platform Strain for an Enhanced Taxadiene Biosynthesis by CRISPR/Cas9.

Paclitaxel is an important diterpenoid commonly used as an anticancer drug. Although the paclitaxel biosynthetic pathway has been mostly revealed, some steps remain to be elucidated. The difficulties in plant transformations and the scarcity of the precursor of paclitaxel, (+)-taxa-4(5), 11(12)-diene (taxadiene), have hindered the full comprehension of paclitaxel biochemistry and, therefore, its production by biotechnological approaches. One solution is to use the budding yeast, Saccharomyces cerevisiae, as a platform to elucidate the paclitaxel biosynthesis. As taxadiene is a diterpenoid, its common precursor, geranylgeranyl pyrophosphate (GGPP), needs to be increased in yeast. In this study, we screened various GGPP synthases (GGPPS) to find the most suitable GGPPS for taxadiene production in yeast. We also optimized the taxadiene production by increasing the flux toward the terpenoid pathway. Finally, to remove selection markers, we integrated the required genes using a CRISPR/Cas9 system in the yeast genome. Our result showed that a titer of 2.02 ± 0.40 mg/L (plasmid) and 0.41 ± 0.06 mg/L (integrated) can be achieved using these strategies. This platform strain can be used to readily test the gene candidates for microbial paclitaxel biosynthesis in the future.

Lipidome association with vascular disease and inflammation in HIV+ Ugandan children.

OBJECTIVE: HIV infection and antiretroviral therapy (ART) have both been linked to dyslipidemia and increased cardiovascular disease (CVD). The relationships among the lipidome, immune activation, and subclinical vascular disease in children with perinatally acquired HIV (PHIV) have not been investigated. METHODS: Serum lipid composition, including 13 lipid classes constituting 850 different lipid species were measured by direct infusion-tandem mass spectrometry in samples from 20 ART-treated PHIV and 20 age-matched and sex-matched HIV- Ugandan children. All participants were between 10 and 18 years of age with no other known active infections. PHIVs had HIV-1 RNA level 50 copies/ml or less. In addition, common carotid artery intima--media thickness (IMT), as well as plasma marker of systemic inflammation (hsCRP, IL6, sTNFRa I), monocyte activation (soluble CD14 and CD163), and T-cell activation (expression of CD38 and HLA-DR on CD4+ and CD8+) were evaluated. RESULTS: Median age (Q1, Q3) of study participants was 13 years (11, 15), 37% were boys, 75% were on an NNRTI-based ART regimen. The concentrations of cholesterol ester, LCER, phosphatidylcholines, and sphingomyelin lipid classes were significantly increased in serum of PHIV compared with HIV (P≤0.04). Biomarkers associated with CVD risk including hsCRP, sCD163, and T-cell activation were directly correlated with lipid species in PHIV (P ≤ 0.04). Contents of free fatty acids including palmitic (16 : 0), stearic (18 : 0), and arachidic acid (20 : 0) were positively correlated with IMT in PHIV. CONCLUSION: Serum lipidome is altered in young virally suppressed PHIV on ART. A direct association between inflammation and lipid species known to be associated with CVD was observed.

Untargeted metabolomics of strawberry (Fragaria x ananassa 'Camarosa') fruit from plants grown under osmotic stress conditions.

BACKGROUND: Plants activate defense mechanisms to cope with adverse environmental conditions, leading to the accumulation and / or depletion of general and specialized metabolites. In this study, a multiplatform untargeted metabolomics strategy was employed to evaluate metabolic changes in strawberry fruit of cv. Camarosa grown under osmotic stress conditions. Liquid chromatography-mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) data from strawberries grown under two water-deficit conditions, irrigated at 95% crop evapotranspiration (ETc) and 85% ETc, and one excess salt condition with a 80 mmol L-1 NaCl solution, were analyzed to determine treatment effects on fruit metabolism. RESULTS: Multivariate principal component analysis, orthogonal projections to latent structures - discriminant analysis (OPLS-DA), and univariate statistical analyses were applied to the data set. While multivariate analyses showed group separation by treatment, T-tests and fold change revealed 12 metabolites differentially accumulated in strawberries from different treatments - among them phenolic compounds, glycerophospholipids, phytosterols, carbohydrates, and an aromatic amino acid. CONCLUSION: Untargeted metabolomic analysis allowed for the annotation of compounds differentially accumulated in strawberry fruit from plants grown under osmotic stress and non-stressed plants. The metabolic disturbance in plants under stress involved metabolites associated with the inhibition of reactive oxygen species and cell-wall and membrane lipid biosynthesis, which might serve as osmotic stress biomarkers. © 2019 Society of Chemical Industry.

Optimized Camellia sinensis var. sinensis, Ilex paraguariensis, and Aspalathus linearis blend presents high antioxidant and antiproliferative activities in a beverage model.

A statistical optimization study was conducted to obtain a tea containing fermented rooibos (Aspalathus linearis), white tea (Camellia sinensis var. sinensis), and roasted mate (Ilex paraguariensis). An optimal combination of these species was proposed. This optimized tea inhibited 64% the lipoperoxidation in vitro and presented a high phenolic content, especially kaempferol, (+)-catechin, (-)-epicatechin, rutin, (-)-epigallocatechin, and (-)-epicatechin-2-O-gallate. Indeed, the antioxidant effect was confirmed by decreasing 30% the reactive oxygen species generation in human hepatoma carcinoma cells (HepG2, 100 and 240 µg/mL). In the cell viability assay, the GI50 for human colorectal adenocarcinoma epithelial cells (Caco-2) was about 547 µg/mL and 481 µg/mL for HepG2. The pasteurization process (65 °C/30 min) did not affect the total phenolic content and antioxidant activity of the optimized tea formulation. The sensory test indicated an acceptability index of 78%, showing that the analytical approach adopted was feasible to develop a phenolic-rich beverage.

Liquid Chromatography with mass spectrometry analysis of mycotoxins in food samples using silica hydride based stationary phases.

Liquid chromatography with mass spectrometry analysis of selected food samples using silica hydride stationary phases allowed for the identification and quantification of common mycotoxins including aflatoxin B1, B2, G1, G2, ochratoxin A, and fumosinin B1. Phenyl and C18 columns showed relatively similar selectivity based on hydrophobicity but the phenyl phase provides an additional mechanism, π-π interaction. The most hydrophobic of the analyzed compounds was more strongly retained on the C18 column and also has fewer unsaturated sites, which limited the interaction with the phenyl phase. Bean, maize, rice, and wheat samples were harvested and stored under conditions conducive to fungal development, and all samples presented toxin contamination exceeding the maximum tolerable limits.

Transcriptional regulatory networks controlling woolliness in peach in response to preharvest gibberellin application and cold storage.

BACKGROUND: Postharvest fruit conservation relies on low temperatures and manipulations of hormone metabolism to maintain sensory properties. Peaches are susceptible to chilling injuries, such as 'woolliness' that is caused by juice loss leading to a 'wooly' fruit texture. Application of gibberellic acid at the initial stages of pit hardening impairs woolliness incidence, however the mechanisms controlling the response remain unknown. We have employed genome wide transcriptional profiling to investigate the effects of gibberellic acid application and cold storage on harvested peaches. RESULTS: Approximately half of the investigated genes exhibited significant differential expression in response to the treatments. Cellular and developmental process gene ontologies were overrepresented among the differentially regulated genes, whereas sequences in cell death and immune response categories were underrepresented. Gene set enrichment demonstrated a predominant role of cold storage in repressing the transcription of genes associated to cell wall metabolism. In contrast, genes involved in hormone responses exhibited a more complex transcriptional response, indicating an extensive network of crosstalk between hormone signaling and low temperatures. Time course transcriptional analyses demonstrate the large contribution of gene expression regulation on the biochemical changes leading to woolliness in peach. CONCLUSION: Overall, our results provide insights on the mechanisms controlling the complex phenotypes associated to postharvest textural changes in peach and suggest that hormone mediated reprogramming previous to pit hardening affects the onset of chilling injuries.

Antidepressant and antistress activity of GC-MS characterized lipophilic extracts of Ginkgo biloba leaves.

Lipophilic extracts of Ginkgo biloba L. leaves were tested for their possible role on rodent models of depression and stress. Lipophilic extracts of Ginkgo leaves (LEG) at (50 and 100 mg/kg, p.o.) exhibited dose dependent, significant antidepressant activity in the behavioral despair test and learned helplessness rodent model of depression. The activities were comparable to that of imipramine (15 mg/kg) and EGb 761 (50 mg/kg). In the cold immobilization stress induced gastric ulcer model of stress, only the LEG showed a significant reduction in the ulcer index. GC-MS characterization of this bioactive extract was found to be rich in a group of 6-alkyl salicylates (6-AS), along with a fatty alcohol, fatty acids and cardanols. The n-heptadecenyl salicylate represented 60% of the 6-AS. Notable was the absence of dihydroxy alkylphenols which are linked to allergic reactions similar to the urushiols present in poison ivy. In commercial products of Ginkgo, these dihydroxy phenols as well as the favorable 6-AS are removed during enrichment of flavonol glycosides and terpenic lactones. The current findings suggest that intact carboxylic acid groups containing 6-AS are the bioactive components of the lipophilic extract of Ginkgo leaves with antidepressant and antistress activities.

An insect parasitoid carrying an ochratoxin producing fungus.

The insect parasitoid Prorops nasuta has been introduced from Africa to many coffee-producing countries in an attempt to control the coffee berry borer. In this paper, we report on the sequencing of the ITS LSU-rDNA and beta-tubulin loci used to identify a fungus isolated from the cuticle of a P. nasuta that emerged from coffee berries infected with the coffee berry borer. The sequences were compared with deposits in GenBank and the fungus was identified as Aspergillus westerdijkiae. The fungus tested positive for ochratoxin A production, with varying levels depending on the media in which it was grown. These results raise the possibility that an insect parasitoid might be disseminating an ochratoxin-producing fungus in coffee plantations.