The Time-Resolved Salt Stress Response of Dunaliella tertiolecta-A Comprehensive System Biology Perspective.

Algae-driven processes, such as direct CO2 fixation into glycerol, provide new routes for sustainable chemical production in synergy with greenhouse gas mitigation. The marine microalgae Dunaliella tertiolecta is reported to accumulate high amounts of intracellular glycerol upon exposure to high salt concentrations. We have conducted a comprehensive, time-resolved systems biology study to decipher the metabolic response of D. tertiolecta up to 24 h under continuous light conditions. Initially, due to a lack of reference sequences required for MS/MS-based protein identification, a high-quality draft genome of D. tertiolecta was generated. Subsequently, a database was designed by combining the genome with transcriptome data obtained before and after salt stress. This database allowed for detection of differentially expressed proteins and identification of phosphorylated proteins, which are involved in the short- and long-term adaptation to salt stress, respectively. Specifically, in the rapid salt adaptation response, proteins linked to the Ca2+ signaling pathway and ion channel proteins were significantly increased. While phosphorylation is key in maintaining ion homeostasis during the rapid adaptation to salt stress, phosphofructokinase is required for long-term adaption. Lacking ß-carotene, synthesis under salt stress conditions might be substituted by the redox-sensitive protein CP12. Furthermore, salt stress induces upregulation of Calvin-Benson cycle-related proteins.

Optimization of Rhodococcus erythropolis JCM3201T Nutrient Media to Improve Biomass, Lipid, and Carotenoid Yield Using Response Surface Methodology.

The oleaginous bacterium Rhodococcus erythropolis JCM3201T offers various unique enzyme capabilities, and it is a potential producer of industrially relevant compounds, such as triacylglycerol and carotenoids. To develop this strain into an efficient production platform, the characterization of the strain's nutritional requirement is necessary. In this work, we investigate its substrate adaptability. Therefore, the strain was cultivated using nine nitrogen and eight carbon sources at a carbon (16 g L-1) and nitrogen (0.16 g L-1) weight ratio of 100:1. The highest biomass accumulation (3.1 ± 0.14 g L-1) was achieved using glucose and ammonium acetate. The highest lipid yield (156.7 ± 23.0 mg g-1DCW) was achieved using glucose and yeast extract after 192 h. In order to enhance the dependent variables: biomass, lipid and carotenoid accumulation after 192 h, for the first time, a central composite design was employed to determine optimal nitrogen and carbon concentrations. Nine different concentrations were tested. The center point was tested in five biological replicates, while all other concentrations were tested in duplicates. While the highest biomass (8.00 ± 0.27 g L-1) was reached at C:N of 18.87 (11 g L-1 carbon, 0.583 g L-1 nitrogen), the highest lipid yield (100.5 ± 4.3 mg g-1DCW) was determined using a medium with 11 g L-1 of carbon and only 0.017 g L-1 of nitrogen. The highest carotenoid yield (0.021 ± 0.001 Abs454nm mg-1DCW) was achieved at a C:N of 12 (6 g L-1 carbon, 0.5 g L-1 nitrogen). The presented results provide new insights into the physiology of R. erythropolis under variable nutritional states, enabling the selection of an optimized media composition for the production of valuable oleochemicals or pigments, such as rare odd-chain fatty acids and monocyclic carotenoids.

Lipopeptides development in cosmetics and pharmaceutical applications: A comprehensive review.

Lipopeptides are surface active, natural products of bacteria, fungi and green-blue algae origin, having diverse structures and functionalities. In analogy, a number of chemical synthesis techniques generated new designer lipopeptides with desirable features and functions. Lipopetides are self-assembly guided, supramolecular compounds which have the capacity of high-density presentation of the functional epitopes at the surface of the nanostructures. This feature contributes to their successful application in several industry sectors, including food, feed, personal care, and pharmaceutics. In this comprehensive review, the novel class of ribosomally synthesized lipopeptides is introduced alongside the more commonly occuring non-ribosomal lipopeptides. We highlight key representatives of the most researched as well as recently described lipopeptide families, with emphasis on structural features, self-assembly and associated functions. The common biological, chemical and hybrid production routes of lipopeptides, including prominent analogues and derivatives are also discussed. Furthermore, genetic engineering strategies aimed at increasing lipopeptide yields, diversity and biological activity are summarized and exemplified. With respect to application, this work mainly details the potential of lipopeptides in personal care and cosmetics industry as cleansing agents, moisturizer, anti-aging/anti-wrinkling, skin whitening and preservative agents as well as the pharmaceutical industry as anitimicrobial agents, vaccines, immunotherapy, and cancer drugs. Given that this review addresses human applications, we conclude on the topic of safety of lipopeptide formulations and their sustainable production.

The potential of biofuels from first to fourth generation.

The steady increase in human population and a rising standard of living heighten global demand for energy. Fossil fuels account for more than three-quarters of energy production, releasing enormous amounts of carbon dioxide (CO2) that drive climate change effects as well as contributing to severe air pollution in many countries. Hence, drastic reduction of CO2 emissions, especially from fossil fuels, is essential to tackle anthropogenic climate change. To reduce CO2 emissions and to cope with the ever-growing demand for energy, it is essential to develop renewable energy sources, of which biofuels will form an important contribution. In this Essay, liquid biofuels from first to fourth generation are discussed in detail alongside their industrial development and policy implications, with a focus on the transport sector as a complementary solution to other environmentally friendly technologies, such as electric cars.

Adaptation of Proteome and Metabolism in Different Haplotypes of Rhodosporidium toruloides during Cu(I) and Cu(II) Stress.

Rhodosporidium toruloides is a carotenogenic, oleogenic yeast that is able to grow in diverse environments. In this study, the proteomic and metabolic responses to copper stress in the two haplotypes IFO0559 and IFO0880 were assessed. 0.5 mM Cu(I) extended the lag phase of both strains significantly, while only a small effect was observed for Cu(II) treatment. Other carotenogenic yeasts such as Rhodotorula mucilaginosa are known to accumulate high amounts of carotenoids as a response to oxidative stress, posed by excess copper ion activity. However, no significant increase in carotenoid accumulation for both haplotypes of R. toruloides after 144 h of 0.5 mM Cu(I) or Cu(II) stress was observed. Yet, an increase in lipid production was detected, when exposed to Cu(II), additionally, proteins related to fatty acid biosynthesis were detected in increased amounts under stress conditions. Proteomic analysis revealed that besides the activation of the enzymatic oxidative stress response, excess copper affected iron-sulfur and zinc-containing proteins and caused proteomic adaptation indicative of copper ion accumulation in the vacuole, mitochondria, and Golgi apparatus.

Effects of Light on Growth and Metabolism of Rhodococcus erythropolis.

Rhodococcus erythropolis is resilient to various stressors. However, the response of R. erythropolis towards light has not been evaluated. In this study, R. erythropolis was exposed to different wavelengths of light. Compared to non-illuminated controls, carotenoid levels were significantly increased in white (standard warm white), green (510 nm) and blue light (470 nm) illuminated cultures. Notably, blue light (455, 425 nm) exhibited anti-microbial effects. Interestingly, cellular lipid composition shifted under light stress, increasing odd chain fatty acids (C15:0, C17:1) cultured under white (standard warm white) and green (510 nm) light. When exposed to blue light (470, 455, 425 nm), fatty acid profiles shifted to more saturated fatty acids (C16:1 to C16:0). Time-resolved proteomics analysis revealed several oxidative stress-related proteins to be upregulated under light illumination.

Oleaginous yeasts- substrate preference and lipid productivity: a view on the performance of microbial lipid producers.

BACKGROUND: Oleaginous yeasts are promising microbial platforms for sustainable, bio-based production of biofuels and oleochemical building blocks. Bio-based residues provide sustainable and cost-effective carbon sources for fermentative yeast oil production without land-use change. Considering the regional abundancy of different waste streams, we chose complex biomass residue streams of marine origin; macroalgae hydrolysate, and terrestrial origin; wheat straw hydrolysate in the presence, and absence of corn steep liquor as a complex nitrogen source. We investigated the biomass and lipid yields of an array of well-described oleaginous yeasts; R. glutinis, T. asahii, R. mucilaginosa, R. toruloides, C. oleaginosus growing on these hydrolysates. Furthermore, their sugar utilization, fatty acid profile, and inhibitory effect of the hydrolysates on yeast growth were compared. For correlative reference, we initially performed comparative growth experiments for the strains on individual monomeric sugars separately. Each of these monomeric sugars was a dominant carbon source in the complex biomass hydrolysates evaluated in this study. In addition, we evaluated N-acetylglucosamine, the monomeric building block of chitin, as a low-cost nitrogen and carbon source in yeast fermentation. RESULTS: C. oleaginosus provided the highest biomass and lipid yields. In the wheat straw and brown algae hydrolysates, this yeast strain gained 7.5 g/L and 3.8 g/L lipids, respectively. Cultivation in algae hydrolysate resulted in a higher level of unsaturated fatty acids in the lipids accumulated by all yeast strains. R. toruloides and C. oleaginosus were able to effectively co-utilize mannitol, glucose, and xylose. Growth rates on wheat straw hydrolysate were enhanced in presence of corn steep liquor. CONCLUSIONS: Among the yeast strains investigated in this study, C. oleaginosus proved to be the most versatile strain in terms of substrate utilization, productivity, and tolerance in the complex media. Various fatty acid profiles obtained on each substrate encourage the manipulation of culture conditions to achieve the desired fatty acid composition for each application. This could be accomplished by combining the element of carbon source with other formerly studied factors such as temperature and oxygen. Moreover, corn steep liquor showed promise for enhancement of growth in the oleaginous strains provided that carbon substrate is available.

Identifying carbohydrate-active enzymes of Cutaneotrichosporon oleaginosus using systems biology.

BACKGROUND: The oleaginous yeast Cutaneotrichosporon oleaginosus represents one of the most promising microbial platforms for resource-efficient and scalable lipid production, with the capacity to accept a wide range of carbohydrates encapsulated in complex biomass waste or lignocellulosic hydrolysates. Currently, data related to molecular aspects of the metabolic utilisation of oligomeric carbohydrates are sparse. In addition, comprehensive proteomic information for C. oleaginosus focusing on carbohydrate metabolism is not available. RESULTS: In this study, we conducted a systematic analysis of carbohydrate intake and utilisation by C. oleaginosus and investigated the influence of different di- and trisaccharide as carbon sources. Changes in the cellular growth and morphology could be observed, depending on the selected carbon source. The greatest changes in morphology were observed in media containing trehalose. A comprehensive proteomic analysis of secreted, cell wall-associated, and cytoplasmatic proteins was performed, which highlighted differences in the composition and quantity of secreted proteins, when grown on different disaccharides. Based on the proteomic data, we performed a relative quantitative analysis of the identified proteins (using glucose as the reference carbon source) and observed carbohydrate-specific protein distributions. When using cellobiose or lactose as the carbon source, we detected three- and five-fold higher diversity in terms of the respective hydrolases released. Furthermore, the analysis of the secreted enzymes enabled identification of the motif with the consensus sequence LALL[LA]L[LA][LA]AAAAAAA as a potential signal peptide. CONCLUSIONS: Relative quantification of spectral intensities from crude proteomic datasets enabled the identification of new enzymes and provided new insights into protein secretion, as well as the molecular mechanisms of carbo-hydrolases involved in the cleavage of the selected carbon oligomers. These insights can help unlock new substrate sources for C. oleaginosus, such as low-cost by-products containing difficult to utilize carbohydrates. In addition, information regarding the carbo-hydrolytic potential of C. oleaginosus facilitates a more precise engineering approach when using targeted genetic approaches. This information could be used to find new and more cost-effective carbon sources for microbial lipid production by the oleaginous yeast C. oleaginosus.

Microbial lipid production by oleaginous yeasts grown on Scenedesmus obtusiusculus microalgae biomass hydrolysate.

Due to increasing oil prices and climate change concerns, biofuels have become increasingly important as potential alternative energy sources. However, the use of arable lands and valuable resources for the production of biofuel feedstock compromises food security and negatively affect the environment. Single cell oils (SCOs), accumulated by oleaginous yeasts, show great promise for efficient production of biofuels. However, the high production costs attributed to feedstocks or raw materials present a major limiting factor. The fermentative conversion of abundant, low-value biomass into microbial oil would alleviate this limitation. Here, we explore the feasibility of utilizing microalgae-based cell residues as feedstock for yeast oil production. We developed an efficient, single-step enzymatic hydrolysis to generate Scenedesmus obtusiusculus hydrolysate (SH) without thermo-chemical pretreatment. With this eco-friendly process, glucose conversion efficiencies reached 90-100%. Cutaneotrichosporon oleaginosus, Cryptococcus curvatus and Rhodosporidium toruloides were cultivated on SH as sole nutrients source. Only C. oleaginosus was able to accumulate intracellular lipids, with a 35% (g lipid/g DCW) content and a yield of 3.6 g/L. Our results demonstrate the potential valorization of algal biomass into desired end-products such as biofuels.

Optimization of protein isolation by proteomic qualification from Cutaneotrichosporon oleaginosus.

In the last decades, microbial oils have been extensively investigated as a renewable platform for biofuel and oleochemical production. Offering a potent alternative to plant-based oils, oleaginous microorganisms have been the target of ongoing metabolic engineering aimed at increasing growth and lipid yields, in addition to specialty fatty acids. Discovery proteomics is an attractive tool for elucidating lipogenesis and identifying metabolic bottlenecks, feedback regulation, and competing biosynthetic pathways. One prominent microbial oil producer is Cutaneotrichosporon oleaginosus, due to its broad feedstock catabolism and high lipid yield. However, this yeast has a recalcitrant cell wall and high cell lipid content, which complicates efficient and unbiased protein extraction for downstream proteomic analysis. Optimization efforts of protein sample preparation from C. oleaginosus in the present study encompasses the comparison of 8 lysis methods, 13 extraction buffers, and 17 purification methods with respect to protein abundance, proteome coverage, applicability, and physiochemical properties (pI, MW, hydrophobicity in addition to COG, and GO analysis). The optimized protocol presented in this work entails a one-step extraction method utilizing an optimal lysis method (liquid homogenization), which is augmented with a superior extraction buffer (50 mM Tris, 8/2 M Urea/Thiourea, and 1% C7BzO), followed by either of 2 advantageous purification methods (hexane/ethanol or TCA/acetone), depending on subsequent applications and target studies. This work presents a significant step forward towards implementation of efficient C. oleaginosus proteome mining for the identification of potential targets for genetic optimization of this yeast to improve lipogenesis and production of specialty lipids. Graphical abstract.

Uremic Serum Induces Inflammation in Cultured Human Endothelial Cells and Triggers Vascular Repair Mechanisms.

Inflammation and cardiovascular disease (CVD) are common in end-stage renal disease (ESRD) patients whose vascular endothelium is in direct contact with the uremic toxins found in the blood. These toxins are believed to affect vascular injury and repair process, which is impaired in ESRD patients. The exact mechanisms behind these interactions are not clear. So, we wanted to investigate what happens at the molecular level of endothelial cells when exposed to uremic serum from ESRD patients with diabetes and/or hypertension and its effect on the expression of molecules associated with vascular injury and repair. Cultured human endothelial cells (ECV304) were incubated in the presence of normal or uremic sera from ESRD patients with diabetes and/or hypertension. The expressions of monocyte chemoattractant protein 1 (MCP-1), vascular endothelial growth factor (VEGF), and stromal cell-derived factor 1 (SDF-1) were investigated in endothelial cells (ECV304) by real-time PCR and ELISA. The expression of MCP-1, VEGF, and SDF-1 was elevated in endothelial cells upon exposure to uremic sera from ESRD patients with diabetes and/or hypertension when compared with cells treated with healthy serum. MCP-1 expression in endothelial cells treated with uremic serum from ESRD patients with hypertension only was significantly increased compared with its expression in other cohorts. Exposure of endothelial cells to uremic serum causes endothelial injury and inflammation characterized by an increase in MCP-1 expression. This injury activates the initiation of vascular repair process in these cells by increasing the expression of VEGF and SDF-1. These molecules can be important biomarkers of chronic kidney disease-associated CVD.

Multi-Factorial-Guided Media Optimization for Enhanced Biomass and Lipid Formation by the Oleaginous Yeast Cutaneotrichosporon oleaginosus.

The non-conventional, oleaginous yeast Cutaneotrichosporon oleaginosus is flagged as an industrial cell factory for generation of oleochemicals and biofuels due to its substrate flexibility and high triglyceride yields. In this study, we employed a computational Response Surface Methodology to guide and streamline the experimental media optimization matrix with 12 nitrogen and 10 carbon sources in order to provide for high biomass and lipid accumulation toward an industrially relevant fermentation process. The resulting data provide new insights into C. oleaginosus physiology under variable nutritional states. Accordingly, the lipid content % (lipid weight/yeast dry weight) is controlled by a defined interplay between carbon and nitrogen. In our experimental setup, the highest biomass (18.4 ± 2.20 g/L) and lipid yield (9 ± 0.34 g/L; 49.74 ± 5.16% g lipid weight/g yeast dry cell weight) were obtained with lactose and yeast extract as carbon and nitrogen sources at an elemental weight ratio of 120:1, respectively. Interestingly, with ammonium salts as a N-source, the intracellularly accumulated triglycerides increasingly contain saturated fatty acids, which provides a new route to generate tailored fatty acid profiles for specific oleochemicals or food applications. Our data indicate that a metabolic ceiling for lipid accumulation in C. oleaginosus is obtained with the correct carbon and nitrogen source mixture.

Correlations between serum inflammatory markers and comorbidities in patients with end-stage renal disease.

OBJECTIVES: Chronic inflammatory processes are common in patients with renal disease, especially those with end-stage renal disease (ESRD), in whom inflammatory markers have been shown to increase with renal function deterioration. ESRD is usually accompanied by other chronic diseases such as hypertension and diabetes. The relationships between ESRD comorbidities and serum levels of inflammatory markers have not yet been fully understood. The aim of this study was to assess serum levels of inflammatory markers in different ESRD cohorts and to investigate the correlations between these inflammatory markers and disease comorbidities. METHODS: A total of 147 patients were grouped according to their comorbid conditions: diabetic only, hypertensive only, diabetic and hypertensive, and neither diabetic nor hypertensive. Serum levels of C-reactive protein (CRP), tumour necrosis factor-alpha (TNF-α), and interleukin-1-beta (IL-1ß) were investigated in different ESRD cohorts by enzyme-linked immunosorbent assay. RESULTS: Serum CRP and TNF-α levels were high in diabetic patients (p = 0.0001), hypertensive patients (p = 0.0001), and those who had both diseases (p = 0.0001), when compared to ESRD patients without these comorbidities. There was no significant change in serum IL-1ß levels between patients with diabetes mellitus and/or hypertension compared to patients who did not have these diseases. CONCLUSIONS: Our results showed that, in ESRD patients, CRP and TNF-α seem to be largely affected by patients' comorbidities, unlike IL-1ß, which might be affected more by the dialysis process even in the absence of comorbidities.

SLC35B4, an Inhibitor of Gluconeogenesis, Responds to Glucose Stimulation and Downregulates Hsp60 among Other Proteins in HepG2 Liver Cell Lines.

SLC35B4, solute receptor for UDP-N-acetylglucosamine and UDP-xylose, is associated with diabetes and predisposing conditions. This study investigated the localization of SLC35B4 and compared the differential expression between a knockdown of SLC35B4 and controls in HepG2. Responsiveness to glucose, expression, and localization were assayed using Western blot and immunostaining. Localization was confirmed using a proximity ligation assay. Two-dimensional (2D) gel electrophoresis and MALDI-TOF were used to identify differentially expressed proteins and pathway analysis was performed. SLC35B4 was increased by 60% upon glucose stimulation and localized in Golgi apparatus and endoplasmic reticulum. Presence of SLC35B4 in the Golgi apparatus suggests its involvement in the biosynthesis of glycoconjugate proteins. Four proteins were markedly under-expressed (Hsp60, HspA8, TUBA1A, and ENO1) and linked to the pathogenesis of diabetes or post-translationally modified by O-GlcNAc. Glucose levels activate SLC35B4 expression. This triggers a downstream effect via Hsp60 and other proteins. We hypothesize that the downstream effect on the proteins is mediated via altering the glycosylation pattern inside liver cells. The downstream cascade ultimately alters the ability of cultured liver cells to inhibit endogenous glucose production, and this could play a role in the association of the above-listed genes with the pathogenesis of diabetes.

Colectomy induces an aldosterone-mediated increase in jejunal glucose uptake in rats.

AIMS: The main function of the colon is water and electrolyte absorption. Total colectomy eliminates this colonic function and may alter the absorptive capacity of the small intestine for nutrients. This study examines the effect of total colectomy on jejunal glucose absorption and investigates the potential role of aldosterone in mediating the alterations in glucose uptake post-colectomy using the aldosterone antagonist spironolactone. MAIN METHODS: Total colectomy with ileo-rectal anastomosis was performed on anesthetized rats. Sham rats were identically handled without colon resection. Two days post-surgery, groups of colectomized rats were injected with either a daily subcutaneous dose of spironolactone or sesame oil for 12days. Body weight changes and food and water intake were measured in all experimental groups. Glucose absorption was measured by in-vivo single pass perfusion in the rat jejunum of control, sham, colectomized, colectomized with spironolactone, and colectomized with sesame oil treatment. Na/K ATPase, SGK1, SGLT1 and GLUT2 expressions were determined in jejunal mucosa in control, colectomized and colectomized/spironolactone injected rats by Western blot analysis. Histological assessment was performed on jejunal sections in control and colectomized groups. KEY FINDINGS: Glucose absorption significantly increased in colectomized rats with an observed increase in Na/K ATPase and SGK1 expression. No significant expression change in SGLT1 and GLUT2 was detected in the jejunum in colectomized rats. Spironolactone, however, significantly decreased the glucose uptake post-colectomy and normalized Na/K ATPase and SGK1 expression. SIGNIFICANCE: Our results suggest that jejunal glucose uptake increases post-colectomy as a possible consequence of an aldosterone-mediated function.