Efficient Bioconversion of High Concentration Phytosterol Microdispersion to 4-Androstene-3,17-Dione (AD) by Mycobacterium sp. B3805.

Low solubility of sterols in aqueous media limits efficient steroid production mediated by biocatalytic microorganisms such as Mycobacterium. Sterol emulsion technologies have been developed with low success rates, largely due to the complexity of generating stable and bioavailable particles. In this study, several aqueous dispersions of sterols in-water of different particle sizes were bioconverted to 4-androstene-3,17-dione (AD) in a solvent-free environment, using a classic microorganism Mycobacterium sp. B3805 as a model system. According to our results, the high concentration (20 g/L) phytosterol dispersions with the smallest particle size tested (370 nm) achieved up to 54% (7.4 g/L) AD production yield in 11 days. Moreover, the use of 0.1 biomass/sterols ratio in a complex bioconversion media containing yeast extract, and a 1:1 glucose/microdispersion ratio in the presence of the surfactant DK-Ester P-160 (HLB16), allowed homogenization and increased microdispersion stability, thus achieving the best results using emulsion technologies to date.

Production and Biotransformation of Phytosterol Microdispersions to Produce 4-Androstene-3,17-Dione.

The current state of knowledge regarding phytosterols biotransformation to produce the steroid intermediate 4-androstene-3,17-dione (AD) shows different technologies. However, the initial concentration of phytosterols in batch cultures is limited due to its low solubility in aqueous media, causing serious difficulties for scaling up because of the low mass transfer. In this chapter, we describe a fermentation method of a phytosterol microdispersion with Mycobacterium sp. B3805 in the context of an integral technology for AD production. The microdispersion generation is based on a patent application that claims the production of an aqueous phytosterol microdispersion with an average size particle of 370 nm, and high stability and solubility in water at high phytosterols concentrations (Harting et al., 2012/US0046254). Our results indicate that up to 20 g/L phytosterols can be biotransformed with this technology allowing a good dispersion and stability of reactants in the fermentation broth.

Rosa Mosqueta Oil Prevents Oxidative Stress and Inflammation through the Upregulation of PPAR-α and NRF2 in C57BL/6J Mice Fed a High-Fat Diet.

Background: Rosa mosqueta (RM) oil is characterized by high concentrations of antioxidants and α-linolenic acid (ALA; 18:3n-3). We have previously demonstrated in male C57BL/6J mice that RM decreases hepatic steatosis, a condition strongly associated with oxidative stress and inflammation.Objective: We studied the molecular mechanisms that underlie the role of RM in preventing high-fat diet (HFD)-induced oxidative stress and inflammation.Methods: Male C57BL/6J mice aged 28 d and weighing 12-14 g were divided into the following groups and fed for 12 wk: control diet (CD; 10% fat, 20% protein, and 70% carbohydrates); CD + RM (1.94 mg ALA ⋅ g body weight-1 ⋅ d-1 administered by oral gavage); HFD (60% fat, 20% protein, and 20% carbohydrates); and HFD + RM. General parameters (body weight, visceral fat, and histology); glucose metabolism [homeostasis model assessment and blood glucose area under the curve (AUC)]; oxidative stress [hepatic nuclear factor (erythroid-derived 2)-like-2 (NRF2) and heme oxygenase 1 (HO-1) concentrations]; and inflammation [hepatic peroxisome proliferator-activated receptor α (PPAR-α) and acyl-coenzyme A oxidase 1 (ACOX1) concentrations, blood tumor necrosis factor α (TNF-α) and interleukin 1ß (IL-1ß) concentrations, and Tnfa and Il1b mRNA expression in liver and visceral adipose tissue] were evaluated.Results: In the HFD + RM mice, the final body weight (24.8 ± 1.1 g) was 19% lower than in the HFD mice (30.6 ± 2.8 g) (P < 0.05). Visceral fat was 34% lower in the HFD + RM mice than in the HFD mice (P < 0.05). The blood glucose AUC was 29% lower and Tnfa and Il1b expression levels were 47% and 59% lower, respectively, in the HFD + RM mice than in the HFD mice (P < 0.05). HFD + RM mice had 40% less hepatic steatosis (P < 0.05) and lower upregulation of PPAR-α (33%), ACOX1 (50%), NRF2 (39%), and HO-1 (68%) protein concentrations than did the HFD mice (P < 0.05).Conclusions: Our findings suggest that RM supplementation prevents the obese phenotype observed in HFD-fed mice by downregulating inflammatory cytokine expression and secretion and stimulating hepatic antioxidant and fatty acid oxidation markers.

Effects of rosa mosqueta oil supplementation in lipogenic markers associated with prevention of liver steatosis.

Rosa mosqueta (RM) oil is rich in α-linolenic acid (ALA) - a precursor of eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), and it has a high antioxidant activity due to its abundant content of tocopherols. Additionally, it has been observed that RM oil administration prevents hepatic steatosis. Thus, the aim of this study was to demonstrate the antilipogenic mechanism related to RM oil administration in a high-fat diet (HFD) fed mice model by evaluating markers associated with the regulation of lipid droplet metabolism (PLIN2, PLIN5 and PPAR-γ), and proteins associated with lipogenesis (FAS and SREBP-1c). C57BL/6J mice were fed either a control diet or a HFD, with and without RM oil supplementation for 12 weeks. The results showed that RM oil supplementation decreases hepatic PLIN2 and PPAR-γ mRNA expression and SREBP-1c, FAS and PLIN2 protein levels, whereas we did not find changes in the level of PLIN5 among the groups. These results suggest that modulation of lipogenic markers could be one of the mechanisms, through which RM oil supplementation prevents the hepatic steatosis induced by HFD consumption in a mice model.

Hypolithic cyanobacteria supported mainly by fog in the coastal range of the Atacama Desert.

The Atacama Desert is one of the driest places on Earth, with an arid core highly adverse to the development of hypolithic cyanobacteria. Previous work has shown that when rain levels fall below ~1 mm per year, colonization of suitable quartz stones falls to virtually zero. Here, we report that along the coast in these arid regions, complex associations of cyanobacteria, archaea, and heterotrophic bacteria inhabit the undersides of translucent quartz stones. Colonization rates in these areas, which receive virtually no rain but mainly fog, are significantly higher than those reported inland in the hyperarid zone at the same latitude. Here, hypolithic colonization rates can be up to 80%, with all quartz rocks over 20 g being colonized. This finding strongly suggests that hypolithic microbial communities thriving in the seaward face of the Coastal Range can survive with fog as the main regular source of moisture. A model is advanced where the development of the hypolithic communities under quartz stones relies on a positive feedback between fog availability and the higher thermal conductivity of the quartz rocks, which results in lower daytime temperatures at the quartz-soil interface microenvironment.

Effect of manganese on the secretion of manganese-peroxidase by the basidiomycete Ceriporiopsis subvermispora.

The ligninolytic machinery of the widely used model fungus Ceriporiopsis subvermispora includes the enzymes manganese-peroxidase (MnP) and laccase (Lcs). In this work the effect of Mn(II) on the secretion of MnP was studied. Cultures grown in the absence of Mn(II) showed high levels of mnp transcripts. However, almost no MnP enzyme was detected in the extracellular medium, either by enzymatic activity assays or Western blot hybridizations. In the corresponding mycelia, immuno-electron microscopy experiments showed high levels of MnP enzyme within intracellular compartments. These results suggest that in addition to its well-known effect on transcription regulation of mnp genes, manganese influences secretion of MnP to the extracellular medium. Experiments carried out in the presence of cycloheximide confirmed that the metal is required to secrete MnP already synthesized and retained within the cell.

Hydroquinone and HO differentially affect the ultrastructure and expression of ligninolytic genes in the basidiomycete Ceriporiopsis subvermispora.

The biodegradation of lignin is a highly oxidative process in which various oxidases and peroxidases play a major role. During lignin decay, the generation of aromatic compounds and reactive oxygen species leads to oxidative stress. In this work, the effect of the oxidative compounds H(2)O(2) and hydroquinone in the ligninolytic fungus Ceriporiopsis subvermispora was studied, both at the ultrastructural and at the transcriptional level. Transmission electron microscopy revealed the presence of microvesicles and extensive cytoplasm degeneration after incubation with hydroquinone, but not with H(2)O(2). Studies of the intracellular redox state of the fungus showed that hydroquinone causes a transient decrease in the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio and an increase in the glutathione-S-transferase mRNA levels. These results suggest that hydroquinone produces oxidative stress in this microorganism. On the other hand, it was observed that hydroquinone, but not H(2)O(2), affects Mn-dependent peroxide and laccase transcripts levels. We propose that the mechanism by which the fungus reacts against oxidative stress contributes to its selectivity toward lignin during wood decay.

Expression of genes encoding laccase and manganese-dependent peroxidase in the fungus Ceriporiopsis subvermispora is mediated by an ACE1-like copper-fist transcription factor.

The effect of copper on the expression of genes encoding the ligninolytic enzymes laccase (lcs) and manganese peroxidase (mnp) in Ceriporiopsis subvermispora was evaluated. This metal increased transcript levels of lcs, mnp1 and mnp2. This finding was not unexpected in the case of lcs, since its promoter contains a putative ACE element. Originally characterized in the yeast Saccharomyces cerevisiae, ACE is the target sequence of the ACE1 copper-responsive transcription factor in this microorganism. Analysis of the promoter regions of mnp genes revealed the presence of formerly unnoticed ACE elements. Based on the ace1 gene from Phanerochaete chrysosporium, we isolated and characterized an ACE1-like transcription factor from C. subvermispora (Cs-ACE1) through complementation of a S. cerevisiae ace1Delta strain. Surprisingly, ACE1 factors from both basidiomycetes exhibit substantial differences, not only structurally but also in their ability to complement the aforementioned yeast strain. Specific binding of Cs-ACE1 to its cognate DNA sequence was confirmed by electrophoretic mobility-shift assays.