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The disposal of waste from industrial sources is becoming a serious problem throughout the world. To produce one liter (L) of ethanol, about 15-L Ethanol Spent Water (ESW) is discharged from the ethanol industry adversely impacting aquatic and terrestrial ecosystems. This study investigated the bioremediation of ESW in two stages, first filtration through biochar (byproduct after HTL processing of algal biomass) followed by bioremediation using algae. In the first step up to 99.4% toxic melanoidin pigment and suspended pollutants from ESW were removed. The colorless ESW was then tested for the growth of marine Parachlorella kessleri-I (PK-I), and freshwater Chlorella vulgaris (CV) and Chlorella sorokiniana-I (CS-I). Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) analysis was conducted before and after bioremediation of ESW using algae. The chemical oxygen demand (COD) 28,900 mg L−1 was reduced to 55–118 mg L−1, biological oxygen demand (BOD) 7200 mg L−1 was reduced to 28–55 mg L−1, total organic carbon (98.7%), phenol (64%), sugar (99.5%), nutrients and toxic metal concentrations were also reduced significantly after 12 days of algae-cultivation in ESW. The highest biomass (115.4 mg L−1 day−1) and lipid (9.1 mg L−1 day−1) productivity were observed in PK-1 using biochar-filtered ESW. The biodiesel properties and cetane number 48.1–63.9 in FAME studied by GC–MS were observed as per the international standard of fuels. The ESW cultivated algal biomass contained a significant amount of lipids, proteins, and carbohydrates compared to the control medium grown biomass. Thus algae-derived biochar has the potential to effectively remediate the hazardous ESW thereby facilitating the efficient growth of different algal species to generate the feedstock for biofuel applications. Bioremediation of raw Ethanol Spent Wash (ESW). About 15-L wastewater generates to produce 1-L ethanol from Ethanol Distillery. The filtration of ESW through algae-based biochar facilitated the growth of marine and freshwater algae as a bioresources for various applications while remediation of wastewater as a circular bioeconomy. [ABSTRACT FROM AUTHOR]

There is increasing evidence that certain Vacuolar protein sorting (Vps) proteins, factors that mediate vesicular protein trafficking, have additional roles in regulating transcription factors at the endosome. We found that yeast mutants lacking the phosphatidylinositol 3-phosphate [PI(3)P] kinase Vps34 or its associated protein kinase Vps15 display multiple phenotypes indicating impaired transcription elongation. These phenotypes include reduced mRNA production from long or G+C-rich coding sequences (CDS) without affecting the associated GAL1 promoter activity, and a reduced rate of RNA polymerase II (Pol II) progression through lacZ CDS in vivo. Consistent with reported genetic interactions with mutations affecting the histone acetyltransferase complex NuA4, vps15Δ and vps34Δ mutations reduce NuA4 occupancy in certain transcribed CDS. vps15Δ and vps34Δ mutants also exhibit impaired localization of the induced GAL1 gene to the nuclear periphery. We found unexpectedly that, similar to known transcription elongation factors, these and several other Vps factors can be cross-linked to the CDS of genes induced by Gcn4 or Gal4 in a manner dependent on transcriptional induction and stimulated by Cdk7/Kin28-dependent phosphorylation of the Pol II C-terminal domain (CTD). We also observed colocalization of a fraction of Vps15-GFP and Vps34-GFP with nuclear pores at nucleus-vacuole (NV) junctions in live cells. These findings suggest that Vps factors enhance the efficiency of transcription elongation in a manner involving their physical proximity to nuclear pores and transcribed chromatin. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The article investigates the role of transmembrane domains and nucleotide-binding domains (NBD) of Cdr1p, a major drug efflux protein of Candida albicans, in drug transport by constructing two sets of protein chimeras. The findings suggest that the chimeric protein could not reach the plasma membrane, probably due to misfolding and subsequent cellular trafficking problems or the rapid degradation of the chimeras. The variant protein comprising either two N-terminal or two C-terminal NBDs of Cdr1 p also resulted in non-functional recombinant strains.

Abstract: Resistance to azole antifungal drugs in clinical isolates of the human fungal pathogen Candida albicans is often caused by constitutive overexpression of the CDR1 gene, which encodes a multidrug efflux pump of the ABC transporter superfamily. To understand the relevance of a recently identified negative regulatory element (NRE) in the CDR1 promoter for the control of CDR1 expression in the clinical scenario, we investigated the effect of mutation or deletion of the NRE on CDR1 expression in two matched pairs of azole-sensitive and resistant clinical isolates of C. albicans. Expression of GFP or lacZ reporter genes from the wild type CDR1 promoter was much higher in the azole-resistant C. albicans isolates than in the azole-susceptible isolates, reflecting the known differences in CDR1 expression in these strains. Deletion or mutation of the NRE resulted in enhanced reporter gene expression in azole-sensitive strains, but did not further increase the already high CDR1 promoter activity in the azole-resistant strains. In agreement with these findings, electrophoretic mobility shift assays showed a reduced binding to the NRE of nuclear extracts from the resistant C. albicans isolates as compared with extracts from the sensitive isolates. These results demonstrate that the NRE is involved in maintaining CDR1 expression at basal levels and that this repression is overcome in azole-resistant clinical C. albicans isolates, resulting in constitutive CDR1 overexpression and concomitant drug resistance. [Copyright &y& Elsevier]

We have earlier shown that transcriptional activation of the Candida drug resistance gene, CDR1, is linked to various stresses wherein a proximal promoter (−345 bp from the transcription start point (TSP)) was found to be predominantly more responsive. In this study we have examined basal expression of the CDR1 proximal promoter by employing a Renilla luciferase reporter system. We observed that upon sequential deletion of the proximal promoter, there was modulation in basal reporter activity. The reporter activity was highest (2.3-fold) in NGY261 (−261 bp from TSP), and was reduced upon subsequent deletions. DNase I footprinting revealed four protected regions (W1, W2, W3 and W4) in the proximal promoter which could represent possible trans-acting factor binding sites and thus might be involved in CDR1 expression. Site-directed mutational analysis of three of these protected regions did not significantly affect the basal reporter activity, however, the mutation of W1 led to a considerable enhancement in reporter activity (∼4-fold) and was designated a negative regulatory element (NRE). Mutation as well as deletion of the W1 sequence in the native promoter (−1147 bp from TSP) and sequential deletion of the 5′-flanking region-harboring W1 (NRE) also resulted in enhanced promoter reporter activity. When the reporter activity of native (NPY1147) and NRE-mutated (NGYM1147) promoter integrants was monitored throughout the growth phase of Candida albicans, there was modulation in reporter activity in both integrants, but interestingly the level of basal reporter activity of the NRE-mutated promoter was always ∼3-fold higher than that of the native promoter. UV cross-linking and affinity purification confirmed that a purified ∼55-kDa nuclear protein specifically interacts with the NRE. Taken together, we have identified a NRE and purified its interactive protein, which may be involved in controlling basal expression of CDR1. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract Plasma membrane (PM) lipid composition imbalances affect drug susceptibilities of the human pathogen Candida albicans. The PM fundamental structure is made up of phospholipid bilayer where phosphatidylethanolamine (PE) contributes as second major phospholipid moieties, which is asymmetrically distributed between the two leaflets of the bilayer. PSD1 and PSD2 genes encode phosphatidylserine decarboxylase which converts phosphatidylserine (PS) into PE in C. albicans cells. Genetic manipulation of PSD1 and PSD2 genes is known to impact virulence, cell wall thickness and mitochondrial function in C. albicans. In the present study, we have examined the impact of PSD1 and PSD2 deletion on physiochemical properties of PM. Our fluorescence recovery after photobleaching (FRAP) experiments point that the PM of psd1 Δ/Δ psd2 Δ/Δ mutant strain displays increased membrane fluidity and reduced PM dipole potential. Further, the result of PSD1 and PSD2 deletion on the thermotropic phase behavior monitored by differential scanning calorimetry (DSC) showed that in comparison to WT, the apparent phase transition temperature is reduced by ~3 °C in the mutant strain. The functional consequence of altered physical state of PM of psd1 Δ/Δ psd2 Δ/Δ mutant strain was evident from observed high diffusion of fluorescent dye rhodamine 6G and radiolabelled fluconazole (FLC). The higher diffusion of FLC resulted in an increased drug accumulation in psd1 Δ/Δ psd2 Δ/Δ mutant cells, which was manifested in an increased susceptibility to azoles. To the best of our knowledge, these results constitute the first report on the effect of the levels of phospholipid biosynthesis enzyme on physiochemical properties of membranes and drug susceptibilities of Candida cells. Graphical abstract Unlabelled Image Highlights • PSD1 and PSD2 genes deletion increase plasma membrane fluidity. • The psd1 Δ/Δ psd2 Δ/Δ mutant shows decreased plasma membrane dipole potential. • The deletion of psd1 Δ/Δ psd2 Δ/Δ results in decrease in phase transition temperature. • Increased drug diffusion and susceptibility are observed in psd1 Δ/Δ psd2 Δ/Δ mutant. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This research work focuses on the potential application of an organic compound, santalol, obtained from santalum album, in the inhibition of the enzyme tyrosinase, which is actively involved in the biosynthesis of melanin pigment. Over-production of melanin causes undesirable pigmentation in humans as well as other organisms and significantly downgrades their aesthetic value. The study is designed to explain the purification of tyrosinase from the mushroom Agaricus bisporus, followed by activity assays and enzyme kinetics to give insight into the santalol-modulated tyrosinase inhibition in a dose-dependent manner. The multi-spectroscopic techniques such as UV-vis, fluorescence, and isothermal calorimetry are employed to deduce the efficiency of santalol as a potential candidate against tyrosinase enzyme activity. Experimental results are further verified by molecular docking. Santalol, derived from the essential oils of santalum album, has been widely used as a remedy for skin disorders and a potion for a fair complexion since ancient times. Based on enzyme kinetics and biophysical characterization, this is the first scientific evidence where santalol inhibits tyrosinase, and santalol may be employed in the agriculture, food, and cosmetic industries to prevent excess melanin formation or browning. [ABSTRACT FROM AUTHOR]

• Bark of populus ciliata of the salicaceae family is traditionally used for blood purification, treatment of rheumatism, fatigue, and menstrual cramps pain. • HPTLC analysis revealed the higher concentrations of gallic acid, salicin, and quercetin in the ethyl acetate fraction of P. ciliata bark. • In-vitro studies revealed the strong antioxidant, antimicrobial, anti-inflammatory, and anticancer properties of the ethyl acetate fraction of P. ciliata bark. • The findings of the current study validate the ethnomedicinal use of bark of P. ciliata. Populus ciliata Wall. ex Royle (Salicaceae family) is a fast-growing, multipurpose native tree of the Himalayas. Traditionally, the tree's bark is used for blood purification, treatment of rheumatism, and fatigue, and to relieve the pain of menstrual cramps. The present study deals with screening crude methanolic extracts and fractions (n-hexane, chloroform, ethyl acetate, and n-butanol) of P. ciliata bark and leaves for total polyphenolic contents, antioxidant, antimicrobial, anti-inflammatory, and cytotoxic activities. Results showed that among all tested samples, the bark ethyl acetate fraction had the highest polyphenolic contents and strong antioxidant potential (DPPH 25.75±3.60 µg/mL; ABTS 33.55±2.96 µg/mL; FRAP 70.39±1.75 µM of Fe equivalents). The HPTLC analysis confirmed the presence of gallic acid, salicin, and quercetin molecules in the extracts and different fractions of P. ciliata whereas ethyl acetate fractions showed their maximum quantity. The ethyl acetate fraction of bark also revealed higher antibacterial activity against Pseudomonas aeruginosa (MIC 0.0156 mg/mL), Klebsiella pneumoniae and Staphylococcus aureus (MIC 0.0312 mg/mL), Bacillus subtilis and Escherichia coli (MIC 0.0625 mg/mL), and antifungal activity against Fusarium oxysporum (MIC 0.125 mg/mL) and Rosellinia necatrix (0.250 mg/mL), whereas butanol fraction of bark showed maximum antifungal activity against Candida auris (MIC 1.25 mg/mL). The highest anti-inflammatory activity was observed in bark ethyl acetate fraction (IC 50 0.113±0.017 mg/mL for egg albumin and 0.160±0.014 mg/mL for bovine serum albumin assay) in comparison with other extracts and fractions. Additionally, bark crude extract and its ethyl acetate fraction were observed more effective against colon cancer cell lines (IC 50 values <0.170 mg/mL for HCT-116 and <0.340 mg/mL for SW-620). The study concludes that the bark ethyl acetate fraction is the most biologically active fraction due to a higher number of polyphenolic contents (salicin, gallic acid, and quercetin) and among both plant parts, bark has a higher medicinal value than leaves. Therefore, this fraction can be further explored to extract biologically active compounds of pharmaceutical, food, and cosmetic values. [ABSTRACT FROM AUTHOR]

Robust microorganisms are required for sustainable second-generation biofuel production. We evaluated the growth and fermentation performance of six natural isolates that were derived from grape wine and medicinal herbs using a wide range of carbon sources, rice and wheat straw hydrolysates as well as stress conditions associated with second-generation ethanol production. Sequence analysis of the 5.8S internal transcribed spacer (ITS) and species-specific PCR amplification of the HO gene region assigned the natural isolates to Saccharomyces cerevisiae . Restriction fragment length polymorphism (RFLP) analysis of the mitochondrial DNA revealed that natural yeast isolates are genetically closer to the laboratory strain BY4741 than to the CEN.PK strains. Dextrose fermentation by a natural isolate, MTCC4780, under semi-anaerobic conditions produced maximum ethanol yields of 0.44 g/g and 0.39 g/g, respectively, with and without the stresses encountered during lignocellulosic ethanol fermentation. However, MTCC4780 produced ethanol yields of 0.48 g/g, 0.42 g/g and 0.45 g/g, respectively, with glucose, rice and wheat straw enzymatic hydrolysate fermentation in a bioreactor. The isolates MTCC4781 and MTCC4796 showed higher growth and fermentation performance than did MTCC4780 in the presence of elevated temperature and pre-treatment inhibitors. Taken together, the MTCC4780, MTCC4781 and MTCC4796 strains have the potential to serve as a platform for lignocellulosic ethanol production under stresses associated with second-generation biofuel production. [ABSTRACT FROM AUTHOR]

In this study, we have specifically blocked a key step of sphingolipid (SL) biosynthesis in Candida glabrata by disruption of the orthologs of ScIpt1 and ScSkn1. Based on their close homology with S. cerevisiae counterparts, the proteins are predicted to catalyze the addition of a phosphorylinositol group onto mannosyl inositolphosphoryl ceramide (MIPC) to form mannosyl diinositolphosphoryl ceramide (M(IP)2C), which accounts for the majority of complex SL structures in S. cerevisiae membranes. High throughput lipidome analysis confirmed the accumulation of MIPC structures in ΔCgipt1 and ΔCgskn1 cells, albeit to lesser extent in the latter. Noticeably, ΔCgipt1 cells showed an increased susceptibility to azoles; however, ΔCgskn1 cells showed no significant changes in the drug susceptibility profiles. Interestingly, the azole susceptible phenotype of ΔCgipt1 cells seems to be independent of the ergosterol content. ΔCgipt1 cells displayed altered lipid homeostasis, increased membrane fluidity as well as high diffusion of radiolabeled fluconazole (3H-FLC), which could together influence the azole susceptibility of C. glabrata. Furthermore, in vivo experiments also confirmed compromised virulence of the ΔCgipt1 strain. Contrarily, specific functions of CgSkn1 remain unclear. [ABSTRACT FROM AUTHOR]

Molasses is one of the most common feedstocks for ethanol production, which requires a large amount of water for dilution during fermentation. In present study, paddy straw hydrolysate (PSH) was integrated to molasses as diluent and fermentation bioprocesses were developed using a robust Saccharomyces cerevisiae NGY10 strain. Batch fermentation in serum bottle using 40% v/v molasses diluted with PSH under optimized conditions at 35 °C produced 97.11 ± 2.36 g/L (12.31% v/v) ethanol with 0.46 g/g yield and 2.2 g/L.h productivity in 30 h. During batch fermentation in a bioreactor at 35 °C and pH 5.5 with aeration (0.3 vvm for initial 6 h) maximum 104.63 ± 2.97 g/L (13.26% v/v) ethanol was produced with 0.47 g/g yield and 4.35 g/L.h productivity in 24 h. In fed-batch fermentation, ∼6.06% higher ethanol titre was achieved than batch fermentation, but productivity was decreased. Remarkably, simultaneous saccharification and fermentation at 40 °C with high solid loading (22.5% w/v) and pre-saccharification produced 104.21 ± 3.23 g/L (13.21% v/v) ethanol in 72 h when 1:1 mixture of molasses and acid-pretreated paddy straw was used. Taken together, the integration of PSH as diluent to molasses not only increases the ethanol yield and productivities but also saves water consumption and operational costs, and can be accommodated in existing sugar mill infrastructures. [Display omitted] • Molasses and paddy straw hydrolysate can be integrated for ethanol production. • 13.26% v/v ethanol was produced with 40% molasses at 35oC in optimized conditions. • S. cerevisiae NGY10 produced maximum ethanol in batch fermentation of molasses. • In SSF at 40oC with 22.5% solid loading, strain NGY10 produced 13.21% v/v ethanol. • Molasses diluted with paddy straw hydrolysate can be used to produce ethanol. [ABSTRACT FROM AUTHOR]

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 chromatographymass 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. [ABSTRACT FROM AUTHOR]

Paf1 complex (Paf1C) is a transcription elongation factor whose recruitment is stimulated by Spt5 and the CDKs Kin28 and Bur1, which phosphorylate the Pol II C-terminal domain (CTD) on Serines 2, 5, and 7. Bur1 promotes Paf1C recruitment by phosphorylating C-terminal repeats (CTRs) in Spt5, and we show that Kin28 enhances Spt5 phosphorylation by promoting Bur1 recruitment. It was unclear, however, whether CTD phosphorylation by Kin28 or Bur1 also stimulates Paf1C recruitment. We find that Paf1C and its Cdc73 subunit bind diphosphorylated CTD repeats (pCTD) and phosphorylated Spt5 CTRs (pCTRs) in vitro, and that cdc73 mutations eliminating both activities reduce Paf1C recruitment in vivo. Phosphomimetic (acidic) substitutions in the Spt5 CTR sustain high-level Paf1C recruitment in otherwise wild-type cells, but not following inactivation of Bur1 or Kin28. Furthermore, inactivating the pCTD/pCTR-interaction domain (PCID) in Cdc73 decreases Paf1C-dependent histone methylation in cells containing non-phosphorylatable Spt5 CTRs. These results identify an Spt5 pCTR-independent pathway of Paf1C recruitment requiring Kin28, Bur1, and the Cdc73 PCID. We propose that pCTD repeats and Spt5 pCTRs provide separate interaction surfaces that cooperate to ensure high-level Paf1C recruitment. [ABSTRACT FROM AUTHOR]

Summary: Kes1, and other oxysterol-binding protein superfamily members, are involved in membrane and lipid trafficking through trans-Golgi network (TGN) and endosomal systems. We demonstrate that Kes1 represents a sterol-regulated antagonist of TGN/endosomal phosphatidylinositol-4-phosphate signaling. This regulation modulates TOR activation by amino acids and dampens gene expression driven by Gcn4, the primary transcriptional activator of the general amino acid control regulon. Kes1-mediated repression of Gcn4 transcription factor activity is characterized by nonproductive Gcn4 binding to its target sequences, involves TGN/endosome-derived sphingolipid signaling, and requires activity of the cyclin-dependent kinase 8 (CDK8) module of the enigmatic “large Mediator” complex. These data describe a pathway by which Kes1 integrates lipid metabolism with TORC1 signaling and nitrogen sensing. [Copyright &y& Elsevier]

In contrast to the deregulated hepatocellular division that is a feature of many hepatic diseases and malignancies, physiologic liver growth during embryonic development and after partial hepatectomy (PH) in adults is characterized by tightly controlled cell proliferation. We used forward genetic screening in zebrafish to test the hypothesis that a similar genetic program governs physiologic liver growth during hepatogenesis and regeneration after PH. We identified the uhrf1 gene, a cell cycle regulator and transcriptional activator of top2a expression, as required for hepatic outgrowth and embryonic survival. By developing a methodology to perform PH on adult zebrafish, we found that liver regeneration in uhrf1+/- adult animals is impaired. uhrf1 transcript levels dramatically increase after PH in both mice, and zebrafish and top2a is not up-regulated in uhrf1+/- livers after PH. This indicates that uhrf1 is required for physiologic liver growth in both embryos and adults and illustrates that zebrafish livers regenerate. [ABSTRACT FROM AUTHOR]

The ATP binding cassette (ABC) transporters, first discovered as high-affinity nutrient importers in bacteria, rose to prominence when their ability to confer multidrug resistance (MDR) to cancer cells was realized. The most characterized human permeability glycoprotein (P-gp) is a dominant exporter of anti-cancer drugs and its overexpression is directly linked to MDR. The overexpression of drug efflux pumps belonging to the ABC superfamily is also a frequent cause of resistance to antifungals. Fungi has a battery of ABC proteins, but in variable numbers and at different subcellular locations. These proteins perform many critical functions, from serving as gatekeepers for xenobiotic cleansing to translocating various structurally unrelated cargoes, including lipids, fatty acids, ions, peptides, sterols, metabolites and toxins. Their emerging additional roles in cellular physiology and virulence call for attention to analyze and re-examine their divergent functions in yeast. In brief, this review traces the history of ABC transporters in yeast and discusses their typical physiological functions that go beyond their well-known role as antifungal drug efflux pumps. [ABSTRACT FROM AUTHOR]

Background: To meet the present transportation demands and solve food versus fuel issue, microbial lipid-derived biofuels are gaining attention worldwide. This study is focussed on high-throughput screening of oleaginous yeast by microwave-aided Nile red spectrofluorimetry and exploring pongamia shell hydrolysate (PSH) as a feedstock for lipid production using novel oleaginous yeast Rhodotorula pacifica INDKK. Results: A new oleaginous yeast R. pacifica INDKK was identified and selected for microbial lipid production. R. pacifica INDKK produced maximum 12.8 ± 0.66 g/L of dry cell weight and 6.78 ± 0.4 g/L of lipid titre after 120 h of growth, showed high tolerance to pre-treatment-derived inhibitors such as 5-hydroxymethyl furfural (5-HMF), (2 g/L), furfural (0.5 g/L) and acetic acid (0.5 g/L), and ability to assimilate C3, C5 and C6 sugars. Interestingly, R. pacifica INDKK showed higher lipid accumulation when grown in alkali-treated saccharified PSH (AS-PSH) (0.058 ± 0.006 g/L/h) as compared to acid-treated detoxified PSH (AD-PSH) (0.037 ± 0.006 g/L/h) and YNB medium (0.055 ± 0.003 g/L/h). The major fatty acid constituents are oleic, palmitic, linoleic and linolenic acids with an estimated cetane number (CN) of about 56.7, indicating the good quality of fuel. Conclusion: These results suggested that PSH and R. pacifica INDKK could be considered as potential feedstock for sustainable biodiesel production. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 has a positive sense RNA genome of 29.9 kb in size, showing high sequence similarity to the BAT-CoV, SARS-CoV, MERS-CoV. SARS-CoV-2 is composed of 14 open reading frames (ORFs), which encodes for a total of 27 proteins divided into structural and non-structural proteins (NSPs). The fundamental structural protein-encoding genes are a spike protein (S) gene, envelope protein (E) gene, a membrane protein (M) gene, and a nucleocapsid protein (N) gene. They make about 33% of the entire genome and are vital for the viral life cycle. Rest 67% is distributed among different NSPs (such as Mpro, helicase, and RNA-dependent RNA polymerase) encoding genes across the ORFs, which are involved in virus-cell receptor interactions during viral entry. Researchers are trying to formulate vaccines, therapeutic antibodies or protein-targeted antiviral drugs to control the spread. This review proceeds stepwise through the COVID-19 outbreak, structural and genomic organization, entry mechanism, pathogenesis, and finally highlighting the essential proteins involved at each step that might be potential targets for drug discovery. Currently, approved treatment modalities consist of only supportive care and oxygen supplementation. This review is established on the current knowledge that has expanded on structural motifs and topology of proteins and their functions. [ABSTRACT FROM AUTHOR]

The present study is an attempt to determine the lipid composition of Candida auris and to highlight if the changes in lipids can be correlated to high drug resistance encountered in C. auris. For this, the comparative lipidomics landscape between drug-susceptible (CBS10913T) and a resistant hospital isolate (NCCPF_470033) of C. auris was determined by employing high throughput mass spectrometry. All major groups of phosphoglycerides (PGL), sphingolipids, sterols, diacylglycerols (DAG) and triacylglycerols (TAG), were quantitated along with their molecular lipid species. Our analyses highlighted several key changes where the NCCPF_470033 showed an increase in PGL content, specifically phosphatidylcholine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, and phosphatidylethanolamine; odd chain containing lipids and accumulation of 16:1-DAG and 16:0-DAG; depletion of 18:1-TAG and 18:0-TAG. The landscape of molecular species displayed a distinct imprint between isolates. For example, the levels of unsaturated PGLs, contributed by both odd and even-chain fatty acyls were higher in resistant NCCPF_470033 isolate, resulting in a higher unsaturation index. Notwithstanding, several commonalities of lipid compositional changes between resistant C. auris and other Candida spp. the study could also identify distinguishable changes in specific lipid species in C. auris. Together, the data highlights the modulation of membrane lipid homeostasis associated with drug-resistant phenotype of C. auris. [ABSTRACT FROM AUTHOR]

Considering the relevance of drug transporters belonging to ABC and MFS superfamilies in pathogenic Candida species, there has always been a need to have an overexpression system where these membrane proteins for functional analysis could be expressed in a homologous background. We could address this unmet need by constructing a highly drug-susceptible Candida glabrata strain deleted in seven dominant ABC transporters genes such as CgSNQ2, CgAUS1, CgCDR1 , CgPDH1 , CgYCF1 , CgYBT1 and CgYOR1 and introduced a GOF mutation in transcription factor (TF) CgPDR1 leading to a hyper-activation of CgCDR1 locus. The expression system was validated by overexpressing four GFP tagged ABC (CgCDR1 , CgPDH1 , CaCDR1 and ScPDR5) and an MFS (CgFLR1) transporters genes facilitated by an engineered expression plasmid to integrate at the CgCDR1 locus. The properly expressed and localized transporters were fully functional, as was revealed by their several-fold increased drug resistance, growth kinetics, localization studies and efflux activities. The present homologous system will facilitate in determining the role of an individual transporter for its substrate specificity, drug efflux, pathogenicity and virulence traits without the interference of other major transporters. [ABSTRACT FROM AUTHOR]

Sphingolipids (SLs) are essential to fungal survival and represent a major class of structural and signaling lipids. Unique SL structures and their biosynthetic enzymes in filamentous fungi make them an ideal drug target. Several studies have contributed towards the functional characterization of specific SL metabolism genes, which have been complemented by advanced lipidomics methods which allow accurate identification and quantification of lipid structures and pathway mapping. These studies have provided a better understanding of SL biosynthesis, degradation and regulation networks in filamentous fungi, which are discussed and elaborated here. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

ATP-binding cassette (ABC) transporters help export various substrates across the cell membrane and significantly contribute to drug resistance. However, a recent study reported an unusual case in which the loss of anABCtransporter in Candida albicans, orf19.4531 (previously named ROA1), increases resistance against antifungal azoles, which was attributed to an altered membrane potential in the mutant strain. To obtain further mechanistic insights into this phenomenon, here we confirmed that the plasma membrane-localized transporter (renamed CDR6/ROA1 for consistency with C. albicans nomenclature) could efflux xenobiotics such as berberine, rhodamine 123, and paraquat. Moreover, a CDR6/ROA1 null mutant, NKKY101, displayed increased susceptibility to these xenobiotics. Interestingly, fluorescence recovery after photobleaching (FRAP) results indicated that NKKY101 mutant cells exhibited increased plasma membrane rigidity, resulting in reduced azole accumulation and contributing to azole resistance. Transcriptional profiling revealed that ribosome biogenesis genes were significantly up-regulated in the NKKY101 mutant. As ribosome biogenesis is a well-known downstream phenomenon of target of rapamycin (TOR1) signaling, we suspected a link between ribosome biogenesis and TOR1 signaling in NKKY101. Therefore, we grew NKKY101 cells on rapamycin and observed TOR1 hyperactivation, which leads to Hsp90-dependent calcineurin stabilization and thereby increased azole resistance. This in vitro finding was supported by in vivo data from a mouse model of systemic infection in which NKKY101 cells led to higher fungal load after fluconazole challenge than wild-type cells. Taken together, our study uncovers a mechanism of azole resistance in C. albicans, involving increased membrane rigidity and TOR signaling. [ABSTRACT FROM AUTHOR]

• Engineering a natural diploid S. cerevisiae for increased FAEE production. • Rational allocation of metabolic fluxes towards expanding FAEE precursor pools. • Process optimization in shake flask increased the titer to ∼40-folds. • Scale-up production in fed-batch bioreactor yielded a final 5.0 g/L FAEE titer. Developing microbial chassis for synthesizing value-added compounds is the most eco-friendly approach to minimize the impacts of overusing fossil fuels and other harsh chemicals. Recent research has focussed on harnessing microbial fatty acid metabolism to generate oleochemicals for use in industries as diverse as fuel production, cosmetics, lubricants and more. Considering the economic viability of generating "green biocatalysts", we reconfigured the fatty acid biosynthesis pathway in an environmentally robust strain of Saccharomyces cerevisiae MTCC4796 to produce fatty acid ethyl ester (FAEE) with high yield and titer. We applied the push–pull-block strategy wherein a heterologous wax ester synthase (WS2) was initially integrated to establish a FAEE biosynthetic pathway (pull) with additional rewiring including overexpression of PDH-bypass pathway genes for increasing the supply of precursor fatty acyl-CoA (push) and single allele disruption of non-essential genes from competing pathways (block) to maximize the metabolic flux towards FAEE overproduction. Our final engineered strain NGYT carrying 11 genetic modifications produced 26 mg/L and 1 g/L of FAEE before and after glucose optimization respectively. This titer was further scaled-up to reach 5 g/L in fed-batch bioreactors, which, to our knowledge, is the highest reported FAEE titer achieved from Saccharomyces cerevisiae with glucose as carbon source. Additionally, the FAEE's produced by NGYT strain showed a higher percentage of monounsaturated fatty acids, which are ideally suitable for biodiesel applications. Given the global emphasis on the development of cleaner fuels, the findings from this study expedite our progress towards industrial scale-FAEE production as a substitute to conventional diesel. [ABSTRACT FROM AUTHOR]

[Display omitted] • The multiple products formation from sugarcane industry waste was assessed. • Undetoxified sugarcane bagasse and molasses were converted into microbial oil. • Maximum lipid titer (11.8 g/L) and β-carotene (210.4 mg/L) was obtained. • Biodiesel produced showed good oxidative stability and cold flow properties. • Xylooligosaccharides production was assessed. Dependency on fossil fuels raises an economic and ecological concern that has urged to look for alternative sources of energy. Bio-refinery concept is one of the alternate frameworks for the biomass conversion into biofuel and other value-added by-products. The present work illustrates importance of an oleaginous yeast Rhodotorula pacifica INDKK in an integrated bio-refinery field by utilizing renewable sugars generated from lignocellulosic biomass. The maximum 11.8 g/L lipid titer, 210.4 mg/L β-carotene and 7.1 g animal feed were produced by R. pacifica INDKK in bioreactor containing 5% (v/v) molasses supplemented with enzymatically hydrolyzed and alkali-pretreated sugarcane bagasse hydrolysate (35% v/v). Furthermore, xylooligosaccharides (20.6 g/L), a beneficial prebiotics were also produced from the hemicellulosic fraction separated after alkali pretreatment of bagasse. This novel concept of integrated yeast bio-refinery for concomitant production of biodiesel and multiple value-added products with minimum waste generation is proposed as a sustainable and profitable process. [ABSTRACT FROM AUTHOR]

Independent studies from our group and others have provided evidence that sphingolipids (SLs) influence the antimycotic susceptibility of Candida species. We analyzed the molecular SL signatures of drug-resistant clinical isolates of Candida auris , which have emerged as a global threat over the last decade. This included Indian hospital isolates of C. auris , which were either resistant to fluconazole (FLCR) or amphotericin B (AmBR) or both drugs. Relative to Candida glabrata and Candida albicans strains, these C. auris isolates were susceptible to SL pathway inhibitors such as myriocin and aureobasidin A, suggesting that SL content may influence azole and AmB susceptibilities. Our analysis of SLs confirmed the presence of 140 SL species within nine major SL classes, namely the sphingoid bases, Cer, αOH-Cer, dhCer, PCer, αOH-PCer, αOH-GlcCer, GlcCer, and IPC. Other than for αOH-GlcCer, most of the SLs were found at higher concentrations in FLCR isolates as compared to the AmBR isolates. SLs were at intermediate levels in FLCR + AmBR isolates. The observed diversity of molecular species of SL classes based on fatty acyl composition was further reflected in their distinct specific imprint, suggesting their influence in drug resistance. Together, the presented data improves our understanding of the dynamics of SL structures, their synthesis, and link to the drug resistance in C. auris. • Candida auris isolates are susceptible to sphingolipid inhibitors myriocin and aureobasidin A. • The distribution of sphingolipid species is distinct among C. auris isolates resistant to different antifungals. • Phytoceramides are the most abundant class of sphingolipid. • Cer(d18:1/18:1) is the major of ceramide species in C. auris. • d19:2 glucosylceramide backbone is typically in abundance in AmB resistant C. auris isolates. [ABSTRACT FROM AUTHOR]

This study focuses on the use of synthetic and low cost carbon sources for enhanced biomass and lipid production from oleaginous yeast strains using batch and fed batch fermentation. Five strains were selected out of 20 strains which were all isolated from various habitats, on the basis of lipid accumulating potential using Sudan B black dye. The selected isolates were characterized and their lipid production was optimized by batch cultivation in waste extract. Gas chromatography analysis revealed that lipids from all isolated yeast strains contained mainly long-chain fatty acids such as oleic acid, palmitic acid, stearic acid and linoleic acid. On the basis of lipid profile and production, two most suitable oleaginous yeast strains were selected for further experiments, i.e. fed batch fermentation using glucose (control) and sugarcane molasses as feed. Using glucose as feed, 38.8 ± 0.9 g/L and 29.4 ± 0.2 g/L biomass dry cell weight (DCW) and total lipids were produced respectively, whereas with sugarcane molasses feed, 50 ± 1 g/L (DCW) biomass and 39.2 ± 0.6 g/L lipids were produced by Rhodosporidium toruloides ATCC204091. After transesterification the lipid profile was analyzed by GC–MS revealing suitability for use as a potential feedstock for biodiesel production. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]