Isolation and identification of carotenoid-producing yeast and evaluation of antimalarial activity of the extracted carotenoid(s) against P. falciparum

Plasmodial resistance to a variety of plant-based antimalarial drugs has led toward the discovery of more effective antimalarial compounds having chemical or biological origin. Since natural compounds are considered as safer drugs, in this study, yeast strains were identified and compared for the production of carotenoids that are well-known antioxidants and this metabolite was tested for its antiparasitic activity. Plasmodium falciparum 3D7 strain was selected as the target parasite for evaluation of antimalarial activity of yeast carotenoids using in vitro studies. Data were analyzed by FACS (fluorescence-activated cell sorter) and counted via gold standard Giemsa-stained smears. The extracted yeast carotenoids showed a profound inhibitory effect at a concentration of 10–3 µg/µl and 10−4 µg/µl when compared to β- carotene as control. SYBR Green1 fluorescent dye was used to confirm the decrease in parasitaemia at given range of concentration. Egress assay results suggested that treated parasite remained stalled at schizont stage with constricted morphology and were darkly stained. Non-toxicity of carotenoids on erythrocytes and on human liver hepatocellular carcinoma cells (HepG2 cells) was shown at a given concentration. This report provides strong evidence for antimalarial effects of extracted yeast carotenoids, which can be produced via a sustainable and cost-effective strategy and may be scaled up for industrial application

Engineering of Saccharomyces cerevisiae as a consolidated bioprocessing host to produce cellulosic ethanol: recent advancements and current challenges

Lignocellulosic biomass, a rich and inexpensive source of fermentable and renewable carbon, is the most abundant material on earth. Microbial bioprocessing of lignocellulosic biomass to produce biofuels (bioethanol, biobutanol, biodiesel) is a sustainable blueprint to reduce our depleting energy reserves and carbon footprint. Saccharomyces cerevisiae, being an excellent industrial ethanologenic organism, is an ideal candidate to engineer as a consolidated bio-processing (CBP) host, a concept that integrates the different steps of cellulosic ethanol production, from hydrolysis of cellulose to glucose and fermentation of glucose to ethanol in one step. Owing to the developments in the field of genetic engineering and sequencing technologies, research in the past two decades have made pivotal achievements to realize CBP enabling yeast suited for industrial applications. However, overcoming major limitations such as incomplete substrate catabolism, low titres of heterologous protein expression, sub-optimal operational conditions and impediment due to toxic inhibitors/by-products accumulation is still challenging. This review focuses on the progress achieved in constructing S. cerevisiae to produce bioethanol in a CBP framework. The different techniques of developing cellulolytic yeast strains are initially explained followed by relevant strategies to tackle the key bottlenecks associated with the process. Additionally, engineering efforts towards designing hemicellulose-derived sugar utilizing yeast strains are discussed

Metabolomic Profiling Revealed Diversion of Cytidinediphosphate-Diacylglycerol and Glycerol Pathway towards Denovo Triacylglycerol Synthesis in Rhodosporidium toruloides

Oleaginous yeast Rhodosporidium toruloides has great biotechnological potential and scientific interest, yet the molecular rationale of its cellular behavior to carbon and nitrogen ratios with concurrent lipid agglomeration remains elusive. Here, metabolomics adaptations of the R. toruloides in response to varying glucose and nitrogen concentrations have been investigated. In preliminary screening we found that 5% glucose (w/v) was optimal for further analysis in Rhodosporidium toruloides 3641. Hereafter, the effect of complementation to increase lipid agglomeration was evaluated with different nitrogen sources and their concentration. The results obtained illustrated that the biomass (13 g/L) and lipid (9.1 g/L) production were maximum on 5% (w/v) glucose and 0.12% (NH4)2SO4. Furthermore, to shed lights on lipid accumulation induced by nitrogen-limitation, we performed metabolomic analysis of the oleaginous yeast R. toruloides 3641. Significant changes were observed in metabolite concentrations by qualitative metabolomics through gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS), which were mapped onto the governing metabolic pathways. Notable finding in this strain concerns glycerol and CDP-DAG metabolism wherein reduced production of glycerol and phospholipids induced a bypass leading to enhanced de-novo triacylglyceride synthesis. Collectively, our findings help in understanding the central carbon metabolism of R. toruloides which may assist in developing rationale metabolic models and engineering efforts in this organism

ABC transportome inventory of human pathogenic yeast Candida glabrata: Phylogenetic and expression analysis.

ATP-binding cassette (ABC) is one of the two major superfamilies of transporters present across the evolutionary scale. ABC superfamily members came to prominence due to their ability to extrude broad spectrum of substrates and to confer multi drug resistance (MDR). Overexpression of some ABC transporters in clinical isolates of Candida species was attributed to the development of MDR phenotypes. Among Candida species, Candida glabrata is an emerging drug resistant species in human fungal infections. A comprehensive analysis of such proteins in C. glabrata is required to untangle their role not only in MDR but also in other biological processes. Bioinformatic analysis of proteins encoded by genome of human pathogenic yeast C. glabrata identified 25 putative ABC protein coding genes. On the basis of phylogenetic analysis, domain organization and nomenclature adopted by the Human Genome Organization (HUGO) scheme, these proteins were categorized into six subfamilies such as Pleiotropic Drug Resistance (PDR)/ABCG, Multi Drug Resistance (MDR)/ABCB, Multi Drug Resistance associated Protein (MRP)/ABCC, Adrenoleukodystrophy protein (ALDp)/ABCD, RNase L Inhibitor (RLI)/ABCE and Elongation Factor 3 (EF3)/ABCF. Among these, only 18 ABC proteins contained transmembrane domains (TMDs) and were grouped as membrane proteins, predominantly belonging to PDR, MDR, MRP, and ALDp subfamilies. A comparative phylogenetic analysis of these ABC proteins with other yeast species revealed their orthologous relationship and pointed towards their conserved functions. Quantitative real time PCR (qRT-PCR) analysis of putative membrane localized ABC protein encoding genes of C. glabrata confirmed their basal expression and showed variable transcriptional response towards antimycotic drugs. This study presents first comprehensive overview of ABC superfamily proteins of a human fungal pathogen C. glabrata, which is expected to provide an important platform for in depth analysis of their physiological relevance in cellular processes and drug resistance

Identification of genome-wide binding sites of heat shock factor 1, Hsf1, under basal conditions in the human pathogenic yeast, Candida albicans

Abstract The master regulator of thermal stress response, Hsf1, is also an essential determinant for viability and virulence in Candida albicans. Our recent studies highlighted that apart from ubiquitous roles of Hsf1 at higher temperatures, it also has myriad non-heat shock responsive roles essential under iron deprivation and drug defense. Here, we further explored its implications in the normal cellular functioning, by profiling its genome-wide occupancy using chromatin immuno-precipitation coupled to high-density tiling arrays under basal and iron deprived conditions. Hsf1 recruitment profiles revealed that it binds to promoters of 660 genes of varied functions, under both the conditions, however, elicited variability in intensity of binding. For instance, Hsf1 binding was observed on several genes of oxidative and osmotic stress response, cell wall integrity, iron homeostasis, mitochondrial, hyphal and multidrug transporters. Additionally, the present study divulged a novel motif under basal conditions comprising, -GTGn3GTGn3GTG- where, Hsf1 displays strong occupancy at significant number of sites on several promoters distinct from the heat induced motif. Hence, by binding to and regulating major chaperones, stress responsive genes and drug resistance regulators, Hsf1 is imperative in regulating various cellular machineries. The current study provides a framework for understanding novel aspects of how Hsf1 coordinates diverse cellular functions

Development of engineered Candida tropicalis strain for efficient corncob-based xylitol-ethanol biorefinery

Abstract Background Xylitol has a wide range of applications in the pharmaceuticals, cosmetic, food and beverage industry. Microbial xylitol production reduces the risk of contamination and is considered as environment friendly and sustainable compared to the chemical method. In this study, random mutagenesis and genetic engineering approaches were employed to develop Candida tropicalis strains with reduced xylitol dehydrogenase (XDH) activity to eliminate co-substrate requirement for corn cob-based xylitol-ethanol biorefinery. Results The results suggest that when pure xylose (10% w/v) was fermented in bioreactor, the Ethyl methane sulfonate (EMS) mutated strain (C. tropicalis K2M) showed 9.2% and XYL2 heterozygous (XYL2/xyl2Δ::FRT) strain (C. tropicalis K21D) showed 16% improvement in xylitol production compared to parental strain (C. tropicalis K2). Furthermore, 1.5-fold improvement (88.62 g/L to 132 g/L) in xylitol production was achieved by C. tropicalis K21D after Response Surface Methodology (RSM) and one factor at a time (OFAT) applied for media component optimization. Finally, corncob hydrolysate was tested for xylitol production in biorefinery mode, which leads to the production of 32.6 g/L xylitol from hemicellulosic fraction, 32.0 g/L ethanol from cellulosic fraction and 13.0 g/L animal feed. Conclusions This work, for the first time, illustrates the potential of C. tropicalis K21D as a microbial cell factory for efficient production of xylitol and ethanol via an integrated biorefinery framework by utilising lignocellulosic biomass with minimum waste generation

Functional Analysis of CaIPT1, a Sphingolipid Biosynthetic Gene Involved in Multidrug Resistance and Morphogenesis of Candida albicans

In the present study we describe the isolation and functional analysis of a sphingolipid biosynthetic gene, IPT1, of Candida albicans. The functional consequence of the disruption of both alleles of IPT1 was confirmed by mass analysis of its sphingolipid composition. The disruption of both alleles or a single allele of IPT1 did not lead to any change in growth phenotype or total sphingolipid, ergosterol, or phospholipid content of the mutant cells. The loss of mannosyl diinositol diphosphoceramide [M(IP)(2)C] in the ipt1 disruptant, however, resulted in increased sensitivity to drugs like 4-nitroquinoline oxide, terbinafine, o-phenanthroline, fluconazole, itraconazole, and ketoconazole. The increase in drug susceptibilities of ipt1 cells was linked to an altered sphingolipid composition, which appeared to be due to the impaired functionality of Cdr1p, a major drug efflux pump of C. albicans that belongs to the ATP binding cassette superfamily. Our confocal and Western blotting results demonstrated that surface localization of green fluorescent protein-tagged Cdr1p was affected in ipt1 disruptant cells. Poor surface localization of Cdr1p resulted in an impaired ability to efflux fluconazole and rhodamine 6G. The effect of mannosyl inositol phosphoceramide accumulation in the ipt1 mutant and the absence of M(IP)(2)C from the ipt1 mutant on the efflux of drug substrates was very selective. The efflux of methotrexate, a specific substrate of CaMdr1p, another major efflux pump of major facilitator superfamily, remained unaffected in ipt1 mutant cells. Interestingly, changes in sphingolipid composition affected the ability of mutant cells to form proper hyphae in various media. Taken together, our results demonstrate that an altered composition of sphingolipid, which is among the major constituents of membrane rafts, affects the drug susceptibilities and morphogenesis of C. albicans

Disrupting Vesicular Trafficking at the Endosome Attenuates Transcriptional Activation by Gcn4▿

The late endosome (MVB) plays a key role in coordinating vesicular transport of proteins between the Golgi complex, vacuole/lysosome, and plasma membrane. We found that deleting multiple genes involved in vesicle fusion at the MVB (class C/D vps mutations) impairs transcriptional activation by Gcn4, a global regulator of amino acid biosynthetic genes, by decreasing the ability of chromatin-bound Gcn4 to stimulate preinitiation complex assembly at the promoter. The functions of hybrid activators with Gal4 or VP16 activation domains are diminished in class D mutants as well, suggesting a broader defect in activation. Class E vps mutations, which impair protein sorting at the MVB, also decrease activation by Gcn4, provided they elicit rapid proteolysis of MVB cargo proteins in the aberrant late endosome. By contrast, specifically impairing endocytic trafficking from the plasma membrane, or vesicular transport to the vacuole, has a smaller effect on Gcn4 function. Thus, it appears that decreasing cargo proteins in the MVB through impaired delivery or enhanced degradation, and not merely the failure to transport cargo properly to the vacuole or downregulate plasma membrane proteins by endocytosis, is required to attenuate substantially transcriptional activation by Gcn4

Evaluation of divergent yeast genera for fermentation-associated stresses and identification of a robust sugarcane distillery waste isolate Saccharomyces cerevisiae NGY10 for lignocellulosic ethanol production in SHF and SSF

Abstract Background Lignocellulosic hydrolysates contain a mixture of hexose (C6)/pentose (C5) sugars and pretreatment-generated inhibitors (furans, weak acids and phenolics). Therefore, robust yeast isolates with characteristics of C6/C5 fermentation and tolerance to pretreatment-derived inhibitors are pre-requisite for efficient lignocellulosic material based biorefineries. Moreover, use of thermotolerant yeast isolates will further reduce cooling cost, contamination during fermentation, and required for developing simultaneous saccharification and fermentation (SSF), simultaneous saccharification and co-fermentation (SScF), and consolidated bio-processing (CBP) strategies. Results In this study, we evaluated thirty-five yeast isolates (belonging to six genera including Saccharomyces, Kluyveromyces, Candida, Scheffersomyces, Ogatea and Wickerhamomyces) for pretreatment-generated inhibitors {furfural, 5-hydroxymethyl furfural (5-HMF) and acetic acid} and thermotolerant phenotypes along with the fermentation performances at 40 °C. Among them, a sugarcane distillery waste isolate, Saccharomyces cerevisiae NGY10 produced maximum 49.77 ± 0.34 g/l and 46.81 ± 21.98 g/l ethanol with the efficiency of 97.39% and 93.54% at 30 °C and 40 °C, respectively, in 24 h using glucose as a carbon source. Furthermore, isolate NGY10 produced 12.25 ± 0.09 g/l and 7.18 ± 0.14 g/l of ethanol with 92.81% and 91.58% efficiency via SHF, and 30.22 g/l and 25.77 g/l ethanol with 86.43% and 73.29% efficiency via SSF using acid- and alkali-pretreated rice straw as carbon sources, respectively, at 40 °C. In addition, isolate NGY10 also produced 92.31 ± 3.39 g/l (11.7% v/v) and 33.66 ± 1.04 g/l (4.26% v/v) ethanol at 40 °C with the yields of 81.49% and 73.87% in the presence of 30% w/v glucose or 4× concentrated acid-pretreated rice straw hydrolysate, respectively. Moreover, isolate NGY10 displayed furfural- (1.5 g/l), 5-HMF (3.0 g/l), acetic acid- (0.2% v/v) and ethanol-(10.0% v/v) tolerant phenotypes. Conclusion A sugarcane distillery waste isolate NGY10 demonstrated high potential for ethanol production, C5 metabolic engineering and developing strategies for SSF, SScF and CBP