Glutathione production by Saccharomyces cerevisiae: current state and perspectives.

Glutathione (L-γ-glutamyl-cysteinyl-glycine, GSH) is a tripeptide synthesized through consecutive enzymatic reactions. Among its several metabolic functions in cells, the main one is the potential to act as an endogenous antioxidant agent. GSH has been the focus of numerous studies not only due to its role in the redox status of biological systems but also due to its biotechnological characteristics. GSH is usually obtained by fermentation and shows a variety of applications by the pharmaceutical and food industry. Therefore, the search for new strategies to improve the production of GSH during fermentation is crucial. This mini review brings together recent papers regarding the principal parameters of the biotechnological production of GSH by Saccharomyces cerevisiae. In this context, aspects, such as the medium composition (amino acids, alternative raw materials) and the use of technological approaches (control of osmotic and pressure conditions, magnetic field (MF) application, fed-batch process) were considered, along with genetic engineering knowledge, trends, and challenges in viable GSH production. KEY POINTS: • Saccharomyces cerevisiae has shown potential for glutathione production. • Improved technological approaches increases glutathione production. • Genetic engineering in Saccharomyces cerevisiae improves glutathione production.

Enzymatic synthesis optimization of isoamyl butyrate from fusel oil

Many food, cosmetic and pharmaceutical industries have increased their interest in short-chain esters due to their flavor properties. From the industrial standpoint, enzyme reactions are the most economical strategy to reach green products with neither toxicity nor damage to human health. Isoamyl butyrate (pear flavor) was synthesized by isoamyl alcohol (a byproduct of alcohol production) and butyric acid with the use of the immobilized lipase Lipozyme TL IM and hexane as solvents. Reaction variables (temperature, butyric acid concentration, isoamyl alcohol:butyric acid molar ratio and enzyme concentration) were investigated in ester conversion (%), concentration (mol L-1) and productivity (mmol ester g-1 mixture . h), by applying a sequential strategy of the Fractional Factorial Design (FFD) and the Central Composite Rotatable Design (CCRD). High isoamyl butyrate conversion of 95.8% was achieved at 24 hours. At 3 hours, the highest isoamyl butyrate concentration (1.64 mol L-1) and productivity (0.19 mmol ester g-1 mixture . h) were obtained under different reaction conditions. Due to high specificity and selectivity of lipases, process parameters of this study and their interaction with the Lipozyme TL IM are fundamental to understand and optimize the system so as to achieve maximum yield to scale up. Results show that fusel oil may be recycled by the green chemistry process proposed by this study.

Cell mass energetic yields of fed-batch culture by Lipomyces starkeyi.

Estimation of the energy capacity of a microbial cell mass on the basis of its lipid content and elemental composition can be used for the comparative evaluation of different microbial sources of biodiesel. Lipomyces starkeyi cell mass concentration reached 94.6 g/L with 37.4% of lipids in a fed-batch process using xylose and urea as substrates. The fatty acid composition of the yeast oil was quite similar to that of palm oil. L. starkeyi converted more than 80% of the energy contained in xylose into cell mass energy yield. The approach used in this study makes it possible to determine the energy of a cell mass by its elemental composition. A heat of combustion (Q c) of 25.7 (kJ/g) was obtained for the cell mass after 142 h of fed-batch cultivation, which represents approximately 56% of the energy content of diesel oil (45.4 kJ/g). The Q c of the triacylglycerols produced was 48.9 (kJ/g), indicating the potential of this oleaginous yeast for biodiesel production. Our work developed here provides a simple and efficient tool for characterization of this cell mass to further our understanding of its use as a feedstock for bioenergy production.

Effect of feeding strategies on lipid production by Lipomyces starkeyi.

The aim of this study was to produce microbial oil from Lipomyces starkeyi DSM 70296 grown in hemicellulose hydrolysate (H-H). Glucose and xylose were used for batch, fed-batch, repeated fed-batch, and continuous cultures, and H-H was tested at continuous culture. The highest cell and lipid concentrations of 85.4 and 41.8g/L, respectively, were obtained using repeated fed-batch strategy. Continuous culture with dilution rate of 0.03h(-1) presented the highest overall cell (0.443g/g) and lipid yields (0.236g/g). At 0.06h(-1) were obtained the highest cell and lipid productivities. Continuous cultivation using H-H at 0.03h(-1) resulted in higher cell productivity than that obtained using glucose:xylose. Gas chromatography analysis of the esterified lipids indicated that the major constituents of this complex are palmitic acid, stearic acid, oleic acid, and linoleic acid with an estimated cetane number (approximately 61) similar to that of palm biodiesel, which is important for biofuel production.

A cost effective fermentative production of glutathione by Saccharomyces cerevisiae with cane molasses and glycerol

This work aimed to evaluate the effect of sugar cane molasses and glycerol on glutathione (GSH) fermentation by Saccharomyces cerevisiae ATCC 7754 in flask culture using response surface methodology. Under optimized conditions (80 g/L of molasses and 60 g/L of glycerol), the highest GSH and biomass concentration achieved were 119.6 mg/L and 25.3 g/L, respectively. Further studies done in 5 L bioreactor resulted 241.3 mg/L GSH after 96 h in batch fermentation without amino acids addition and the concentration of biomass was 12.1 g/L. In batch fermentation with the addition of the three amino acids (4 mM cysteine, glycine and glutamic acid at 32 h), biomass reached to 25 g/L and GSH, 236.1 mg/L at 96 h of fermentation. The strategy of precursor amino acids addition is a key aspect in increasing the synthesis of GSH.

Optimization of lipid production by the oleaginous yeast Lipomyces starkeyi by random mutagenesis coupled to cerulenin screening.

In this work we performed assays for the genetic improvement of the oleaginous yeast Lipomyces starkeyi DSM 70296 focusing on its utilization for lipid biosynthesis from renewable sources. The genetic optimization was carried out by random mutagenesis by ultraviolet irradiation and mutant selection by cerulenin, a compound displaying inhibitory effects on lipid biosynthesis. Mutants demonstrating normal growth in presence of cerulenin were considered as good candidates for further studies. Using this strategy, we selected 6 mutants for further studies, in which their productivities were evaluated by fermentation in shaken flasks and bioreactor. The evaluation of the fermentative performance of mutants was carried out using xylose as sole carbon source; the fermentation of wild-type strain was used as reference. Using this strategy it was possible to identify one mutant (termed A1) presenting a significant increase in the productivity rates of both biomass and lipid in comparison to wild-type strain. A1 mutant was further studied in bioreactor using the same fermentation parameters optimized for L. starkeyi lipid production from a mixed carbon source (xylose:glucose), as previously determined by other studies in our laboratory. A1 presented a productivity increase of 15.1% in biomass and 30.7% in lipid productivity when compared to the wild-type strain with a similar fatty acid composition, despite a slight increase (approx. 7%) on the unsaturated fraction. Our work demonstrates the feasibility of the random mutagenesis strategy coupled with mutant selection based on cerulenin screening for the genetic improvement of the oleaginous yeast L. starkeyi.