Characterization of cellular development and fatty acid accumulation in thraustochytrid Aurantiochytrium strains of Taiwan

Long-chain saturated and polyunsaturated fatty acids of two new thraustochytrid isolates cultured from Taiwan mangroves, Aurantiochytrium sp. IMB169 and Aurantiochytrium sp. IMB171, were characterized through their cell growth and development in relation to their intracellular lipid accumulation using transmission electron microscopy. Flow cytometry in combination with the lipophilic fluorescent dye BODIPY 505/515 was used to stain and characterize intracellular lipid bodies in the two isolates. The transmission electron microscopy and flow cytometry analyses revealed a progressive accumulation of lipid products in IMB169 and IMB171. Further, selective BODIPY stained cells were successfully separated and enriched using flow cytometry at single cell level. Among the two isolates, IMB169 was found to produce a high level of docosahexaenoic acid. The qualitative and analytical results obtained using electron microscopy and flow cytometry studies were validated by gas chromatography (GC). In addition, a quantitative baseline was established using cell growth, flow cytometry and GC analyses for developing an efficient bioprocessing methodology to selectively enrich thraustochytrids phenotypes with desirable characteristics.

Microaerobic Digestion of Low-Biodegradable Sewage Sludge: Effect of Air Dosing in Batch Reactors

The adoption of prolonged solid retention times during the biological treatment of urban wastewaters is a well-known strategy to reduce sewage sludge production. However, it also results in the production of a biological sludge with low percentages of biodegradable organic matter, also characterized by high humification degrees, which may hamper the anaerobic digestion treatment aimed at sludge stabilization. To accelerate the hydrolytic stage, the application of microaerobic conditions during the anaerobic digestion of low-biodegradable sewage sludge was investigated in this study. In particular, six bio-methanation tests of a real sewage sludge were carried out, introducing air in the bioreactors with doses ranging between 0 and 16.83 L air/kg VSin d, in order to evaluate the air dosage that optimizes the biomethane production and organic matter degradation. Notably, the lower air loading rates investigated in this study, such as 0.68 and 1.37 L air/kg VSin d, led to an increase in methane production of up to 19%, due to a higher degradation of total lipids and proteins. In addition, these microaerobic conditions also resulted in a decrease in the sludge humification degree and in lower volatile fatty acid accumulation.

Function of ORFC of the polyketide synthase gene cluster on fatty acid accumulation in Schizochytrium limacinum SR21

Background As a potential source of polyunsaturated fatty acids (PUFA), Schizochytrium sp. has been widely used in industry for PUFA production. Polyketide synthase (PKS) cluster is supposed to be the primary way of PUFA synthesis in Schizochytrium sp. As one of three open reading frames (ORF) in the PKS cluster, ORFC plays an essential role in fatty acid biosynthesis. However, the function of domains in ORFC in the fatty acid synthesis of Schizochytrium sp. remained unclear. Results In this study, heterologous expression and overexpression were carried out to study the role of ORFC and its domains in fatty acid accumulation. Firstly, ORFC was heterologously expressed in yeast which increased the PUFA content significantly. Then, the dehydratase (DH) and enoyl reductase (ER) domains located on ORFC were overexpressed in Schizochytrium limacinum SR21, respectively. Fatty acids profile analysis showed that the contents of PUFA and saturated fatty acid were increased in the DH and ER overexpression strains, respectively. This indicated that the DH and ER domains played distinct roles in lipid accumulation. Metabolic and transcriptomic analysis revealed that the pentose phosphate pathway and triacylglycerol biosynthesis were enhanced, while the tricarboxylic acid cycle and fatty acids oxidation were weakened in DH-overexpression strain. However, the opposite effect was found in the ER-overexpression strain. Conclusion Therefore, ORFC was required for the biosynthesis of fatty acid. The DH domain played a crucial role in PUFA synthesis, whereas the ER domain might be related to saturated fatty acids (SFA) synthesis in Schizochytrium limacinum SR21. This research explored the role of ORFC in the PKS gene cluster in Schizochytrium limacinum and provided potential genetic modification strategies for improving lipid production and regulating PUFA and SFA content.

Genes Involved in Lipid Metabolism in Coconut

Coconut palm (Cocos nucifera L) is an economically important monocot plant grown in tropical and subtropical regions. Coconut oil is stored in a solid endosperm and has 47.48–50.5% fatty acid component as lauric acid (C12:0). Present research showed that acyl-acyl carrier protein thioesterases (FatA/B) and lysophosphatidic acid acyltransferase (LAAPT) are key enzymes determining medium-chain fatty acid accumulation in coconut oil. Among five CnFatB genes, CnFatB3 expressed specifically in endosperm and in vitro experiment showed that this gene made mainly lauric acid (C12:0) and tetradecenoic acid (C14:1). Overexpression of CnFatB3 in Arabidopsis increased the amounts of C12:0 and C14:0 in transgenic plant. CnLPAAT gene that is expressed specifically in coconut endosperm showed a preference for using acyl-CoAs containing C10:0, C12:0, and C14:0 acyl groups as acyl-donor substrates. Coconut and oil palm are closely related species with approximately 50% lauric acid (C12:0) in their endosperm. The two species have a close evolutionary relationship between predominant gene isoforms and high conservation of gene expression bias in the lipid metabolism pathways. Moreover, since no stable transformation system has been constructed in coconut palm, gene function validations have been done in vitro, or genes transformed into a heterologous system.

Quantitative proteomic comparison of salt stress in Chlamydomonas reinhardtii and the snow alga Chlamydomonas nivalis reveals mechanisms for salt-triggered fatty acid accumulation via reallocation of carbon resources

Background Chlamydomonas reinhardtii is a model green alga strain for molecular studies; its fully sequenced genome has enabled omic-based analyses that have been applied to better understand its metabolic responses to stress. Here, we characterised physiological and proteomic changes between a low-starch C. reinhardtii strain and the snow alga Chlamydomonas nivalis, to reveal insights into their contrasting responses to salinity stress. Results Each strain was grown in conditions tailored to their growth requirements to encourage maximal fatty acid (as a proxy measure of lipid) production, with internal controls to allow comparison points. In 0.2 M NaCl, C. nivalis accumulates carbohydrates up to 10.4% DCW at 80 h, and fatty acids up to 52.0% dry cell weight (DCW) over 12 days, however, C. reinhardtii does not show fatty acid accumulation over time, and shows limited carbohydrate accumulation up to 5.5% DCW. Analysis of the C. nivalis fatty acid profiles showed that salt stress improved the biofuel qualities over time. Photosynthesis and respiration rates are reduced in C. reinhardtii relative to C. nivalis in response to 0.2 M NaCl. De novo sequencing and homology matching was used in conjunction with iTRAQ-based quantitative analysis to identify and relatively quantify proteomic alterations in cells exposed to salt stress. There were abundance differences in proteins associated with stress, photosynthesis, carbohydrate and lipid metabolism proteins. In terms of lipid synthesis, salt stress induced an increase in dihydrolipoyl dehydrogenase in C. nivalis (1.1-fold change), whilst levels in C. reinhardtii remained unaffected; this enzyme is involved in acetyl CoA production and has been linked to TAG accumulation in microalgae. In salt-stressed C. nivalis there were decreases in the abundance of UDP-sulfoquinovose (− 1.77-fold change), which is involved in sulfoquinovosyl diacylglycerol metabolism, and in citrate synthase (− 2.7-fold change), also involved in the TCA cycle. Decreases in these enzymes have been shown to lead to increased TAG production as fatty acid biosynthesis is favoured. Data are available via ProteomeXchange with identifier PXD018148. Conclusions These differences in protein abundance have given greater understanding of the mechanism by which salt stress promotes fatty acid accumulation in the un-sequenced microalga C. nivalis as it switches to a non-growth state, whereas C. reinhardtii does not have this response.

Suppression of Adenosine Deaminase and Xanthine Oxidase Activities by Mineralocorticoid and Glucocorticoid Receptor Blockades Restores Renal Antioxidative Barrier in Oral Contraceptive-Treated Dam

Objective. We tested the hypothesis that postpartum combined oral contraceptive (COC) treatment would induce oxidative stress via the adenosine deaminase-xanthine oxidase pathway in the kidney. We also sought to determine whether mineralocorticoid receptor (MR) or glucocorticoid receptor (GR ) blockade would suppress the activities of ADA and xanthine oxidase caused by postpartum COC treatment in the kidney. Methods. Twenty-four Wistar dams were randomly assigned to 4 groups ( n = 6 / group ). Dams received vehicle (po), COC (1.0 μg ethinylestradiol and 5.0 μg levonorgestrel; po), COC with GR blockade (mifepristone; 80.0 mg/kg; po), and COC with MR blockade (spironolactone; 0.25 mg/kg; po) daily between 3rd and 11th week postpartum. Results. Data showed that postpartum COC caused increased plasma creatinine and urea, increased renal triglyceride/high-density lipoprotein ratio, free fatty acid accumulation, alanine aminotransferase, gamma-glutamyltransferase, uric acid, and activities of renal XO and ADA. On the other hand, postpartum COC resulted in decreased plasma albumin, renal glutathione, and Na+-K+-ATPase activity with no effect on lactate production. However, MR or GR blockade ameliorated the alterations induced by postpartum COC treatment. The present results demonstrate that MR or GR blockade ameliorates postpartum COC-induced increased activities of ADA and xanthine oxidase and restores glutathione-dependent antioxidative defense. Conclusion. These findings implicate the involvements of GR and MR in renal dysfunctions caused by COC in dams via disrupted glutathione antioxidative barrier.

Overexpression of Avocado (Persea americana Mill.) PaRAP2.1 Promotes Fatty Acid Accumulation in Arabidopsis thaliana

Fatty acids in avocado fruit (Persea americana Mill.) are vital composition affecting flavour and nutritive value. Hence, horticulturalists are interested in illustrating the functions of transcription factors on fatty acid accumulation in avocado fruit. In the present study, the APETALA2/ethylene-responsive transcription factor gene, PaRAP2.1, was cloned from avocado mesocarp, and the subcellular localization demonstrated that PaRAP2.1 was located in the cytoplasm and nucleus. The PaRAP2.1 was introduced into Arabidopsis thaliana by Agrobacterium-mediated transformation. Furthermore, PaRAP2.1 were functionally verified its effect on fatty acid biosynthesis. Histological analyses of lipid droplets displayed that the striking difference in the lipid droplets in the mature seeds between PaRAP2.1-overexpressing transgenic and wild-type Arabidopsis thaliana lines were revealed based on confocal microscopy images. Subsequently, fatty acid analyses of PaRAP2.1-overexpressing Arabidopsis thaliana lines displayed the significantly higher contents of fatty acids than those in the wild-type plants. Meanwhile, expression amount of ten genes involving in fatty acid biosynthesis dramatically up-regulated in the mature seeds of PaRAP2.1-overexpressing lines than those of wild-type plants. These results provide a theoretical basis for future research in regard to the function of PaRAP2.1 on fatty acid biosynthesis.

Fatty acid accumulation in feeding types of a natural freshwater fish population

Fatty acids are widely used to study trophic interactions in food web assemblages. Generally, it is assumed that there is a very small modification of fatty acids from one trophic step to another, making them suitable as trophic biomarkers. However, recent literature provides evidence that many fishes possess genes encoding enzymes with a role in bioconversion, thus the capability for bioconversion might be more widespread than previously assumed. Nonetheless, empirical evidence for biosynthesis occurring in natural populations remains scarce. In this study, we investigated different feeding types of perch (Perca fluviatilis) that are specialized on specific resources with different levels of highly unsaturated fatty acids (HUFAs), and analyzed the change between HUFA proportions in perch muscle tissue compared to their resources. Perch showed matching levels to their resources for EPA, but ARA and especially DHA were accumulated. Compound-specific stable isotope analyses helped us to identify the origin of HUFA carbon. Our results suggest that perch obtain a substantial amount of DHA via bioconversion when feeding on DHA-poor benthic resources. Thus, our data indicate the capability of bioconversion of HUFAs in a natural freshwater fish population.