Computational assessment of lipid production in Rhodosporidium toruloides in two-stage and one-stage batch bioprocesses.

Oleaginous yeasts are promising platforms for microbial lipids production as a renewable and sustainable alternative to vegetable oils in biodiesel production. In this paper, a thorough in silico assessment of lipid production in batch cultivation by Rhodosporidium toruloides was developed. By means of dynamic flux balance analysis, the traditional two-stage bioprocess (TSB) performed by the native strain was contrasted with one-stage bioprocess (OSB) using four designed strains obtained by gene knockout strategies. Lipid titer, yield, content, and productivity were analyzed at different initial C/N ratios as relevant performance indicators used in bioprocesses. By weighting these indicators, a global lipid efficiency metric (GLEM) was defined to consider different scenarios. Under simulated conditions, designed strains for lipid overproduction in OSB outperformed the TSB in terms of lipid title (up to threefold), lipid yield (up to 2.4-fold), lipid content (up to 2.8-fold, with a maximum of 76%), and productivity (up to 1.3-fold), depending on C/N ratios. Using these efficiency parameters and the proposed GLEM, the process of selecting the most suitable candidates for lipid production could be carried out before experimental assays. This methodology holds the potential to be extended to other oleaginous microorganisms and diverse strain design techniques.

A new and effective approach to boron removal by using novel boron-specific fungi isolated from boron mining wastewater.

Boron-resistant fungi were isolated from the wastewater of a boron mine in Turkey. Boron removal efficiencies of Penicillium crustosum and Rhodotorula mucilaginosa were detected in different media compositions. Minimal Salt Medium (MSM) and two different waste media containing molasses (WM-1) or whey + molasses (WM-2) were tested to make this process cost effective when scaled up. Both isolates achieved high boron removal yields at the highest boron concentrations tested in MSM and WM-1. The maximum boron removal yield by P. crustosum was 45.68% at 33.95 mg l(-1) initial boron concentration in MSM, and was 38.97% at 42.76 mg l(-1) boron for R. mucilaginosa, which seemed to offer an economically feasible method of removing boron from the effluents.

Assessment of ß-carotene content, cell physiology and morphology of the yellow yeast Rhodotorula glutinis mutant 400A15 using flow cytometry.

Flow cytometry was used to assess ß-carotene content, cell membrane permeability, cell size and granularity in Rhodotorula glutinis mutant 400A15 grown under different oxygen transfer coefficients (k L a) and carbon to nitrogen ratios (C/N). A Doehlert distribution was used in order to select the best conditions that induced the highest carotenoids production. The highest ß-carotene content (0.79 mg g(-1) DCW) at the lowest k L a and C/N (5 × 10(-3) s(-1) and 11.3 respectively). Under these conditions, the biomass concentration attained 18.60 g L(-1). The highest ratio of cells with permeabilised membranes (2.6 %), and the highest cell size and granularity were also obtained under these conditions. It was observed that C/N showed a stronger influence than the k L a on the measured cell parameters.

Extremotolerant fungi as genetic resources for biotechnology.

Increased stress tolerance of economically important plants and microorganisms can improve yields in agriculture and industrial microbiology. The pool of resources used for the genetic modification of crops and industrial fungal strains in the past has been relatively limited, and has frequently included only stress-sensitive organisms. However, certain groups of fungi have evolved specialized mechanisms that enable them to thrive under even the most extreme of environmental conditions. These species can be considered as promising sources of biotechnologically interesting genes. Together with a powerful and convenient high-throughput functional screening method, extremotolerant fungi represent a new opportunity for the identification of stress-tolerance-conferring genes. The approaches described here should provide important contributions to the enhancing of the properties of economically important organisms in the future.

Genetic resources of extremotolerant fungi: a method for identification of genes conferring stress tolerance.

Fungal species from extreme environments represent an underexploited source of stress-resistance genes. These genes have the potential to improve stress tolerance of economically important microorganisms and crops. An efficient high-throughput method for the identification of biotechnologically interesting genes of extremotolerant fungi was developed by constructing a cDNA expression library in Saccharomyces cerevisiae and screening for gain-of-function transformants under stress conditions. The advantages and possible modifications of this method are discussed, and its efficiency is demonstrated using the stress-tolerant basidiomycetous yeast Rhodotorula mucilaginosa. Twelve R. mucilaginosa genes are described that increase halotolerance in S. cerevisiae. These include genes encoding a phosphoglucomutase and a phosphomannomutase. All 12 investigated genes might be useful for the improvement of halotolerance in genetically modified crops or industrial microorganisms.

[Assessment of the MSP-PCR technique for the molecular characterization of Rhodotorula mucilaginosa isolates from northwestern Patagonia]. / Evaluación de la técnica de MSP-PCR para la caracterización molecular de aislamientos de Rhodotorula mucilaginosa provenientes de la Patagonia noroccidental.

The rapid identification of environmental or clinical yeast isolates is important for biodiversity studies and the detection of probable pathogens. Rhodotorula mucilaginosa is a ubiquitous and pigmented yeast capable of infecting immunocompromised patients. In this study, we evaluated the Micro/mini satellite-primed PCR (MSP-PCR) fingerprinting method for the characterization and identification of R. mucilaginosa isolates from natural environments in northwestern Patagonia. There were selected 110 putative R. mucilaginosa isolates from 200 environmental pigmented yeast isolates on the basis of phenotypic criteria. (GTG)5, (GAC)5 and M13 primers were initially evaluated in representative R. mucilaginosa isolates. (GTG)5 allowed a good grouping of these isolates and, at the same time, a good differentiation among closely related species, and thus was selected for subsequent studies. R. mucilaginosa isolates (87%) presented similar (> 60%) MSP-PCR profiles to those of the reference strain CBS 316T. The MSP-PCR technique was effective, both, for the characterization and identification of a large number of R. mucilaginosa environmental isolates as well as for the detection of polymorphisms within the species.

Evaluación de la técnica de MSP-PCR para la caracterización molecular de aislamientos de Rhodotorula mucilaginosa provenientes de la Patagonia noroccidental / Assessment of the MSP-PCR technique for the molecular characterization of Rhodotorula mucilaginosa isolates from northwestern Patagonia

La identificación rápida de levaduras de origen ambiental o clínico es de importancia para el estudio de la biodiversidad de estos microorganismos y para la detección de posibles patógenos. Rhodotorula mucilaginosa es una levadura ubicua y pigmentada, capaz de producir infecciones en pacientes inmunocomprometidos. En este trabajo se evaluó la utilidad de la técnica de fingerprinting conocida como MSP-PCR (Micro/Minisatellite-Primed PCR) en la caracterización e identificación de aislamientos ambientales de R. mucilaginosa provenientes de la Patagonia noroccidental. Sobre la base de sus caracteres fenotípicos, de un total de 200 levaduras pigmentadas se seleccionaron 110 aislamientos que presuntamente corresponderían a la especie R. mucilaginosa. Se evaluaron los iniciadores (GTG)5, (GAC)5 y M13 en aislamientos representativos, y se seleccionó el iniciador (GTG)5 por ser el que permitió una mejor agrupación de los aislamientos pertenecientes a R. mucilaginosa y una mejor diferenciación de éstos con los de especies filogenéticamente próximas. Utilizando dicho iniciador, el 87% de los aislamientos de R. mucilaginosa presentó un perfil de MSP-PCR similar (> 60%) al de la cepa de referencia CBS 316T de R. mucilaginosa. La técnica de MSP-PCR resultó efectiva, tanto para caracterizar e identificar un número elevado de aislamientos ambientales de R. mucilaginosa como para detectar polimorfismos en la especie.

The rapid identification of environmental or clinical yeast isolates is important for biodiversity studies and the detection of probable pathogens. Rhodotorula mucilaginosa is a ubiquitous and pigmented yeast capable of infecting immunocompromised patients. In this study, we evaluated the Micro/mini satellite-primed PCR (MSP-PCR) fingerprinting method for the characterization and identification of R. mucilaginosa isolates from natural environments in northwestern Patagonia. There were selected 110 putative R. mucilaginosa isolates from 200 environmental pigmented yeast isolates on the basis of phenotypic criteria. (GTG)5, (GAC)5 and M13 primers were initially evaluated in representative R. mucilaginosa isolates. (GTG)5 allowed a good grouping of these isolates and, at the same time, a good differentiation among closely related species, and thus was selected for subsequent studies. R. mucilaginosa isolates (87%) presented similar (> 60%) MSP-PCR profiles to those of the reference strain CBS 316T. The MSP-PCR technique was effective, both, for the characterization and identification of a large number of R. mucilaginosa environmental isolates as well as for the detection of polymorphisms within the species.