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

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.

Enhanced biodiesel and ß-carotene production in Rhodotorula pacifica INDKK using sugarcane bagasse and molasses by an integrated biorefinery framework.

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 ß-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.

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.

Nanoporous Sodium Carboxymethyl Cellulose-g-poly (Sodium Acrylate)/FeCl3 Hydrogel Beads: Synthesis and Characterization.

Novel sodium carboxymethyl cellulose-g-poly (sodium acrylate)/Ferric chloride (CMC-g-PNaA/FeCl3) nanoporous hydrogel beads were prepared based on the ionic cross-linking between CMC-g-PNaA and FeCl3. The structure of CMC and CMC-g-PNaA were elucidated by Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR) spectroscopy, and the elemental composition was analyzed by energy dispersive X-ray analysis (EDX). The physicochemical properties of the CMC-g-PNaA/FeCl3 hydrogel beads were analyzed by X-ray diffraction (XRD), scanning electron microscopy (SEM), atomic force microscopy (AFM) and thermogravimetric analysis (TGA). The swelling percentage of hydrogel beads was studied at different time periods. The obtained CMC-g-PNaA/FeCl3 hydrogel beads exhibited a higher nanoporous morphology than those of CMC-g-PNaA and CMC beads. Furthermore, an AFM image of the CMC-g-PNaA/FeCl3 beads shows granule type topology. Compared to the CMC-g-PNaA (189 °C), CMC-g-PNaA/FeCl3 hydrogel beads exhibited improvement in thermal stability (199 °C). Furthermore, CMC-g-PNaA/FeCl3 hydrogel beads depicted a higher swelling percentage capacity of around 1452%, as compared to CMC-g-PNaA (1096%). Moreover, this strategy with preliminary results could be useful for the development of polysaccharide-based hybrid hydrogel beads for various potential applications.

Harnessing pongamia shell hydrolysate for triacylglycerol agglomeration by novel oleaginous yeast Rhodotorula pacifica INDKK.

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.

Chemical profile and therapeutic potentials of Xylocarpus moluccensis (Lam.) M. Roem.: A literature-based review.

ETHNOPHARMACOLOGICAL RELEVANCE: Historically, mangrove plants are among the potential sources of foods and remedies for humans living in the forests and nearby communities. Xylocarpus moluccensis (Lam.) M. Roem., an important mangrove medicinal plant, has been traditionally used for many purposes such as treatment of fever, dysentery, diarrhea, swelling, and abdominal disorders. The aim of the present work was to summarize the chemical reports and biological activities of the mangrove medicinal plant X. moluccensis based on information collected from different databases. MATERIALS AND METHODS: An up-to-date search (till Aug 2019) was carried out in databases such as PubMed, Science Direct, Google Scholar, and various patient offices (e.g., WIPO, CIPO, USPTO) using the keywords: 'Xylocarpus moluccensis', and/or paired with 'ethnobotanical use', and 'phytochemical'. In vitro, ex vivo, or in vivo studies were included. RESULTS: Findings suggest that X. moluccensis contains various important minerals and phytochemicals, where flavonoids, terpenes and terpenoids are the most prominent isolated phyto-constituents of X. moluccensis. Extracts/fractions or isolated compounds from this plant possess diverse biological activities, including anti-inflammatory, anti-microbial, antineoplastic, anti-diarrheal, insecticidal, anti-feedant, neuropharmacological (e.g., central nervous system depressant), anti-atherosclerotic, and lipid-lowering activity. Only one report suggests that the methanol and aqueous extracts of this plant did not exert cytotoxic effects on normal mouse fibroblast cells. However, no clinical studies were reported. CONCLUSIONS: Taken all together, X. moluccensis may be one of the best sources of pharmacologically active lead compounds. More research, however, is necessary to establish the safety and efficacy, and its toxicogenetic effects in animal models.

Redox responsive xylan-SS-curcumin prodrug nanoparticles for dual drug delivery in cancer therapy.

Chemotherapeutic agents with different anticancer mechanisms could enhance therapeutic effect in cancer therapy by their combined application. In this study, redox-sensitive prodrug nanoparticles based on Xyl-SS-Cur conjugate were developed for co-delivery of curcumin and 5-FU in cancer therapy. The Xyl-SS-Cur conjugate was synthesized via covalent conjugation of curcumin to xylan through a disulphide (-S-S-) linkage. The Xyl-SS-Cur conjugate could self-assemble in aqueous medium into nanoparticles and the lipophilic 5-fluorouracil-stearic acid (5-FUSA) prodrug was encapsulated into the hydrophobic core of Xyl-SS-Cur NPs through dialysis membrane method. The obtained Xyl-SS-Cur/5-FUSA NPs had an appropriate size (∼217 ±â€¯2.52 nm), high drug loading of curcumin (∼ 31.4 wt%) and 5-FUSA (∼ 11.8 wt%) and high stability. The interaction of Xyl-SS-Cur/5-FUSA NPs with blood components was investigated by hemolysis study. The cytotoxicity study demonstrated that Xyl-SS-Cur/5-FUSA NPs induced higher cytotoxicity than free drugs against the Human colorectal cancer cells (HT-29, HCT-15). These results indicate that Xyl-SS-Cur/5-FUSA NPs can serve as a promising drug delivery system in cancer therapy.

Production of Oleaginous Organisms or Lipids Using Sewage Water and Industrial Wastewater.

Worldwide, wastewater produced from sewage and industry poses a serious risk to the surrounding environment. As a way to address this problem, an integrated approach for cultivation of oleaginous microorganisms on wastewater leading to effective removal of hazardous components and sustainable production of biodiesel is proposed. Oleaginous yeasts have the unique ability to utilize wastewater as feedstock and accumulate large amounts of triacylglycerols within their cellular compartments at stationary phase (144 h). The lipids stored in an oleaginous microbe can be visualized by fluorescence microscopy and converted into biodiesel through transesterification after extraction. Here, we describe the batch cultivation of oleaginous yeast on sewage and industrial wastewater at 25 °C. High lipid accumulation with efficient removal of toxic chemicals can be achieved by utilizing this integrated method.

Lipophilic 5-fluorouracil prodrug encapsulated xylan-stearic acid conjugates nanoparticles for colon cancer therapy.

In this study, self-assembled nanoparticles based on amphiphilic xylan-stearic acid (Xyl-SA) conjugates have been developed for the efficient delivery of 5-fluorouracil (5-FU) in cancer therapy. The self-assembled behavior of Xyl-SA conjugates in aqueous medium was investigated using pyrene as fluorescent probe. To enhance the loading efficacy of 5-FU, the lipophilic 5-fluorouracil-stearic acid (5-FUSA) prodrug was synthesized and subsequently encapsulated into the hydrophobic core of Xyl-SA NPs. The obtained Xyl-SA/5-FUSA NPs had an appropriate size (~278 nm), high drug loading of 5-FUSA (~14.6 wt%) and high physiological stability. The interaction of the Xyl-SA/5-FUSA NPs with blood components was investigated by hemolysis study. The cell cytotoxic studies demonstrated that Xyl-SA/5-FUSA NPs induced higher cytotoxicity than free drugs against the Human colorectal cancer cells (HT-29, HCT-15). These results indicate that Xyl-SA/5-FUSA NPs can serve as a promising drug delivery system for the efficient delivery of 5-FU in cancer therapy.

Aromatic hydrocarbon biodegradation activates neutral lipid biosynthesis in oleaginous yeast.

In this study, the biodegradation ability of oleaginous yeast Cryptococcus psychrotolerans IITRFD for aromatic hydrocarbons (AHs) was investigated. It was found to completely degrade range of AHs such as 1 g/L phenol, 0.75 g/L naphthalene, 0.50 g/L anthracene and 0.50 g/L pyrene with lipid productivity (g/L/h) of 0.0444, 0.0441, 0.0394 and 0.0383, respectively. This work demonstrated the ring cleavage pathways of AHs by this yeast which follow ortho route for phenol and naphthalene while meta route for anthracene and pyrene degradation. The end products generated during biodegradation of AHs are feed as precursors for de novo triacylglycerols (TAG) biosynthesis pathway of oleaginous yeast. A high quantity of lipid content (46.54%) was observed on phenol as compared to lipid content on naphthalene (46.38%), anthracene (44.97%) and pyrene (44.16%). The lipid profile revealed by GC-MS analysis shows elevated monounsaturated fatty acid (MUFA) content with improved biodiesel quality.

Amaranth seeds (Amaranthus palmeri L.) as novel feedstock for biodiesel production by oleaginous yeast.

The potential of lipid accumulation by oleaginous yeast Cryptococcus vishniaccii grown on amaranth seed aqueous extract (AAE) media was assessed. Maximum cell biomass productivity of 104 mg/L/h, lipid productivity of 54 mg/L/h, and lipid content of 52.31% were recorded on AAE when carbon to nitrogen (C:N) ratio increased from 134 to 147 after removal of ammonia nitrogen. The lipid droplet (LD) size (2.32 ± 0.38 µm) was visualized by fluorescence microscopy using Nile red stain indicating maximum accumulated triacylglycerol (TAG) at C:N 147. Fatty acid methyl ester (FAME) profile obtained after transesterification of extracted lipid revealed the presence of palmitic acid (16:0), palmitoleic acid (16:1), stearic acid (18:0), oleic acid (18:1), and linoleic acid (18:2). Data showed the presence of high monounsaturated fatty acid (MUFA) content (68.17%) depicting improved winter operating conditions of biodiesel. Various quality parameters of biodiesel were evaluated and compared to the American and European biodiesel standards specifications. Based on the lipid productivity, distribution of fatty acids, and evaluated properties obtained; the lipid accumulation by C. vishniaccii utilizing amaranth seeds as substrate could serve as a feasible feedstock for biodiesel production.

Fostering triacylglycerol accumulation in novel oleaginous yeast Cryptococcus psychrotolerans IITRFD utilizing groundnut shell for improved biodiesel production.

The investigation was carried out to examine the potential of triacylglycerol (TAG) accumulation by novel oleaginous yeast isolate Cryptococcus psychrotolerans IITRFD on utilizing groundnut shell acid hydrolysate (GSH) as cost-effective medium. The maximum biomass productivity and lipid productivity of 0.095±0.008g/L/h and 0.044±0.005g/L/h, respectively with lipid content 46% was recorded on GSH. Fatty acid methyl ester (FAME) profile obtained by GC-MS analysis revealed oleic acid (37.8%), palmitic (29.4%) and linoleic (32.8%) as major fatty acids representing balance between oxidative stability (OS) and cold flow filter properties (CFFP) for improved biodiesel quality. The biodiesel property calculated were correlated well with the fuel standards limits of ASTM D6751, EN 14214 and IS 15607. The present findings raise the possibility of using agricultural waste groundnut shell as a substrate for production of biodiesel by novel oleaginous yeast isolate C. psychrotolerans IITRFD.

Converting paper mill sludge into neutral lipids by oleaginous yeast Cryptococcus vishniaccii for biodiesel production.

Paper mill sludge (PMS) was assessed as cheap renewable lignocellulosic biomass for lipid production by the oleaginous yeast Cryptococcus vishniaccii (MTCC 232). The sonicated paper mill sludge extract (PMSE) exhibited enhanced lipid yield and lipid content 7.8±0.57g/l, 53.40% in comparison to 5.5±0.8g/l, 40.44% glucose synthetic medium, respectively. The accumulated triglycerides (TAG) inside the lipid droplets (LDs) were converted to biodiesel by transesterification and thoroughly characterized using GC-MS technique. The fatty acid methyl ester (FAME) profile obtained reveals elevated content of oleic acid followed by palmitic acid, linoleic acid and stearic acid with improved oxidative stability related to biodiesel quality.

Boosting accumulation of neutral lipids in Rhodosporidium kratochvilovae HIMPA1 grown on hemp (Cannabis sativa Linn) seed aqueous extract as feedstock for biodiesel production.

Hemp seeds aqueous extract (HSAE) was used as cheap renewable feedstocks to grow novel oleaginous yeast Rhodosporidium kratochvilovae HIMPA1 isolated from Himalayan permafrost soil. The yeast showed boosted triglyceride (TAG) accumulation in the lipid droplets (LDs) which were transesterified to biodiesel. The sonicated HSAE prepared lacked toxic inhibitors and showed enhanced total lipid content and lipid yield 55.56%, 8.39±0.57g/l in comparison to 41.92%, 6.2±0.8g/l from industrially used glucose synthetic medium, respectively. Supersized LDs (5.95±1.02µm) accumulated maximum TAG in sonicated HSAE grown cells were visualized by fluorescent BODIPY (505/515nm) stain. GC-MS analysis revealed unique longer carbon chain FAME profile containing Arachidic acid (C20:0) 5%, Behenic acid (C22:0) 9.7%, Heptacosanoic acid (C27:0) 14.98%, for the first time in this yeast when grown on industrially competent sonicated HSAE, showing more similarity to algal oils.

CTLA-4 A49G gene polymorphism is not associated with vitiligo in South Indian population.

BACKGROUND: Vitiligo or leukoderma is a chronic skin condition that causes loss of pigment due to destruction of melanocytes, resulting in irregular pale patches of skin. Vitiligo is a polygenic disease and is associated with autoimmunity with an unknown etiology. AIMS: One of the candidate genes which has a strong association with several autoimmune diseases is CTLA-4 gene located in chromosome 2q33 region. We investigated the possible association between CTLA-4 gene polymorphism in exon 1 (A49G) and vitiligo in patients from South India and compared the distribution of this polymorphism to matched control groups. PATIENTS AND METHODS: The polymorphism was detected by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method in 175 patients and 180 normal, age/ethnicity matched individuals. Consistency of genotype frequencies with the Hardy-Weinberg equilibrium was tested using a chi(2) test. RESULTS: There was no significant difference between the genotype (P = 0.93) and allele (P = 0.615) frequencies of CTLA-4 A49G polymorphism in patients and normal healthy individuals. However there was significant association of the CTLA-4 genotype (P = 0.02) and allelic frequency (P = 0.008) between the segmental and non-segmental sub groups within vitiligo. CONCLUSION: Our results indicate that there is no association between CTLA-4 A49G gene polymorphism and vitiligo in southern Indian population.

Correlation and Identification of Variable number of Tandem repeats of eNOS Gene in Coronary artery disease (CAD).

Endothelium-derived nitric oxide (NO) is synthesized from l-arginine by endothelial nitric oxide synthase (eNOS) encoded by the NOS3 gene on chromosome7. Since reduced NO synthesis has been implicated in the development of coronary atherosclerosis; polymorphisms of NOS gene might be associated with increased susceptibility to coronary artery disease (CAD). We therefore undertook this study to determine the association between the occurrence of CAD and eNOS4 b/a polymorphism in South Indian patients. We investigated the polymorphisms in the 27 base-pair tandem repeats in intron4 of the eNOS gene in 100 unrelated CAD patients with positive coronary angiograms and 100 age and sex matched control subjects without any history of symptomatic CAD. The eNOS gene intron4 b/a VNTR polymorphism was analyzed by polymerase chain reaction. The plasma lipids levels and other risk factors were also determined. The genotype frequencies for eNOS4b/b, eNOS4a/b and eNOS4a/a were 63, 26 and 11 per cent in CAD subjects, and 72, 20 and 8 per cent in control subjects, respectively. The genotype frequencies did not differ significantly between the two groups. The frequency of the a allele was 0.24 per cent in CAD subjects and 0.18 per cent in control subjects and no significant association was found between patients and control group (P = 0.57, Odds ratio = 3.62). Plasma lipids, glucose and creatinine levels were significantly increased in CAD group. The genotypic frequencies and the allele frequency did not differ significantly between the CAD patients and controls indicating that this polymorphism was not an independent risk factor for the development of CAD in South Indian patients.