Bioproduction of yeast single cell oil with acute oral toxicity study intended for edible oil application.

Human nutrition and health rely on edible oils. Global demand for edible oils is expanding, necessitating the discovery of new natural oil sources subjected to adequate quality and safety evaluation. However, in contrast to other agricultural products, India's edible oil supply is surprisingly dependent on imports. The microbial oil is generated by fermentation of oleaginous yeast Rhodotorula mucilaginosa IIPL32 MTCC 25056 using biodiesel plant byproduct crude glycerol as a fermentable carbon source. Enriched with monounsaturated fatty acid, nutritional indices mapping based on the fatty acid composition of the yeast SCO, suggested its plausible use as an edible oil blend. In the present study, acute toxicity evaluation of the yeast SCO in C57BL/6 mice has been performed by randomly dividing the animals into 5 groups with 50, 300, 2000, and 5000 mg/Kg yeast SCO dosage, respectively, and predicted the median lethal dose (LD50). Detailed blood biochemistry and kidney and liver histopathology analyses were also reported. The functions of the liver enzymes were also evaluated to check and confirm the anticipated toxicity. To determine cell viability and in vitro biocompatibility, the 3T3-L1 cell line and haemolysis tests were performed. The results suggested the plausible use of yeast SCO as an edible oil blend due to its non-toxic nature in mice models.

A comprehensive characterization of novel CYP-BM3 homolog (CYP-BA) from Bacillus aryabhattai.

Functionalizing C-H bond poses one of the most significant challenges for chemists providing them with very few substrate-specific synthetic routes. Despite being incredibly plastic in their enzymatic ability, they are confined with deficient enzymatic action and limited explicitness of the substrates. In this study, we have endeavored to characterize novel cytochrome P450 from Bacillus aryabhattai (CYP-BA), a homolog of CYP P450-BM3, by taking interdisciplinary approaches. We conducted structure and sequence comparison to understand the conservation pattern for active site residues, conserved fold, evolutionary relationships among others. Molecular dynamics simulations were performed to understand the dynamic nature and interaction with the substrates. CYP-BA was successfully cloned, purified, and characterized. The enzyme's stability toward various physicochemical parameters was evaluated by UV-vis spectroscopy and Circular Dichroism (CD) spectroscopy. Various saturated fatty acids being the natural cytochrome P450 substrates were evaluated as catalytic efficiency of substrate oxidation by CYP-BA. The binding affinity of these natural substrates was monitored against CYP-BA by isothermal titration calorimetry (ITC). The catalytic performance of CYP-BA was satisfactory enough to proceed to the next step, that is, engineering to expand the substrate range to include polycyclic aromatic hydrocarbons (PAH). This is the first evidence of cloning, purifying and characterizing a novel homolog of CYP-BM3 to enable a better understanding of this novel biocatalyst and to provide a platform toward expanding its catalytic process through enzyme engineering.

Scale-up strategy for yeast single cell oil production for Rhodotorula mucilagenosa IIPL32 from corn cob derived pentosan.

This work was aimed to strategically scale-up the yeast lipid production process using Reynolds number as a standard rheological parameter from 50 mL to 50 L scale. Oleaginous yeast Rhodotorula mucilaginosa IIPL32 was cultivated in xylose rich corncob hydrolysate. The fermentation process for growth and maturation was operated in fed-batch with two different C/N ratios of 40 and 60. The hydrodynamic parameters were used to standardize and represent the effect of rheology on the fermentation process. The growth pattern of the yeast was found similar in both shake flask and fermenter with the maximum growth observed at 48 h. The lipid yield increased from 0.4 g/L and 0.5 g/L to 1.3 g/L and 1.83 g/L for 50 mL to 50 L for C/N ratio 40 and 60 respectively. The increase in productivity during the growth phase and lipid accumulation during the maturation phase showed that the scale-up strategy was successful.

Xylitol Production from Lignocellulosic Pentosans: A Rational Strain Engineering Approach toward a Multiproduct Biorefinery.

Kluyveromyces marxianus IIPE453 can utilize biomass-derived fermentable sugars for xylitol and ethanol fermentation. In this study, the xylitol production in the native strain was improved by overexpression of endogenous d-xylose reductase gene. A suitable expression cassette harboring the gene of interest was constructed and incorporated in the native yeast. qPCR analysis demonstrated the 2.1-fold enhancement in d-xylose reductase transcript levels in the modified strain with 1.62-fold enhancement in overall xylitol yield without affecting its ethanol fermenting capacity. Material balance analysis on 2 kg of sugar cane bagasse-derived fermentable sugars illustrated an excess of 58.62 ± 0.15 g of xylitol production by transformed strain in comparison to the wild variety with similar ethanol yield. The modified strain can be suitably used as a single biocatalyst for multiproduct biorefinery application.