Understanding xylose isomerase from Burkholderia cenocepacia: insights into structure and functionality for ethanol production.

The inability of the yeast Saccharomyces cerevisiae to produce ethanol from xylose has hampered the biofuel production from lignocellulosic biomass. However, prior studies reveal that functional expression of xylose isomerase (XI) from Burkholderia cenocepacia (XylABc) in S. cerevisiae has remarkably improved xylose consumption and ethanol productivity. Yet, little is known about kinetic and structural properties of this enzyme. Hereby, a purified recombinant XylA was assayed in vitro, showing optimal enzyme activity at 37 °C and pH 7.2. The Km of XylA for D-xylose was at least threefold lower than the Km results for any XI published to date (e.g. XylA from Piromyces sp.). In addition, oligomerization behavior as a tetramer was observed for XylA in solution. Functional and structural comparative analyses amongst three microbial XIs were further performed as theoretical models, showing that xylose orientation at the active site was highly conserved among the XIs. Mg2+ ions anchor the sugar and guide its pyranoside oxygen towards a histidine residue present at the active site, allowing an acid-base reaction, linearizing xylose. Electrostatic surface analyses showed that small variations in the net charge distribution and dipole moment could directly affect the way the substrate interacts with the protein, thus altering its kinetic properties. Accordingly, in silico modeling suggested the tetramer may be the major functional form. These analyses and the resulting model promote a better understanding of this protein family and pave the way to further protein engineering and application of XylA in the ethanol industry.

γ-decalactone production by Yarrowia lipolytica and Lindnera saturnus in crude glycerol.

Flavor compounds are commonly obtained from chemical synthesis or extracted from plants. These sources have disadvantages, such as racemic mixture generation, more steps to yield the final product, low yield, and high cost, making the microbial fermentation an alternative and potential way to obtain flavor compounds. The most important lactone for flavor application is γ-decalactone, which has an aroma of peach and can be obtained by ricinoleic acid biotransformation through yeast peroxisomal ß-oxidation. The aim of this work was to use crude glycerol, a residual biodiesel industry, for the production of bioaroma from two different yeasts. Yarrowia lipolytica CCMA 0357 and Lindnera saturnus CCMA 0243 were grown at different concentrations (10, 20, and 30% w/v) of substrates (castor oil and crude glycerol) for γ-decalactone production. L. saturnus CCMA 0243 produced higher concentration of y-decalactone (5.8 g/L) in crude glycerol, whereas Y. lipolytica CCMA 0357 showed a maximum production in castor oil (3.5 g/L). Crude glycerol showed better results for γ-decalactone production when compared to castor oil. L. saturnus CCMA 0243 has been shown to have a high potential for γ-decalactone production from crude glycerol.

Rhamnolipids in perspective: gene regulatory pathways, metabolic engineering, production and technological forecasting.

Rhamnolipids have emerged as a very promising class of biosurfactants in the last decades, exhibiting properties of great interest in several industrial applications, and have represented a suitable alternative to chemically-synthesized surfactants. This class of biosurfactants has been extensively studied in recent years, aiming at their large-scale production based on renewable resources, which still require high financial costs. Development of non-pathogenic, high-producing strains has been the focus of a number of studies involving heterologous microbial hosts as platforms. However, the intricate gene regulation network controlling rhamnolipid biosynthesis represents a challenge to metabolic engineering and remains to be further understood and explored. This article provides an overview of the biosynthetic pathways and the main gene regulatory factors involved in rhamnolipid production within Pseudomonas aeruginosa, the prototypal producing species. In addition, we provide a perspective view into the main strategies applied to metabolic engineering and biotechnological production.

Evaluation of heavy metal toxicity in eukaryotes using a simple functional assay.

Although carcinogenesis caused by metals has been intensively investigated, the mechanisms of action, especially at the molecular level, are still unclear. This work aimed to investigate Cd(2+), Cu(2+), Ni(2+), Cr(3+), and Zn(2+) mutagenicity and its relationship with oxidative stress. We have applied the Functional Assay for the Separation of Alleles in Yeast (FASAY) with only minor modifications to detect p53 defects caused by metals. In this method, human p53-coding gene (TP53) expressed in Saccharomyces cerevisiae activates transcription of the ADE2 reporter gene. Yeast cells, expressing p53, were exposed to increased concentrations of metals and, then, plated on media supplemented or not with adenine. Yeast colonies containing functional p53 grow independently of adenine supplementation and colonies containing nonfunctional p53 are dependent on this nutrient. Mutations in the TP53 are implicated in the pathogenesis of half of all human tumors. According to our results, Cd(2+) was found to be the most toxic metal and produced the highest oxidative damage to lipids and proteins. At low concentrations (40 µM), this metal decreased viability and completely inhibited cell growth, while higher concentrations were necessary to produce the same toxic effect by Cu(2+), Cr(3+), and Ni(2+). Zn(2+) showed no significant toxicity. Cd(2+) strongly induced damages and altered the function of p53, while Cu(2+), followed by Cr(3+), showed lower percentages of p53-mutant colonies. Our results point towards a relationship between the loss of functional p53 protein and oxidative stress, a mechanism that can be associated with tumor formation induced by heavy metals in mammalian cells. By this adaptation of FASAY developed by us it is possible to easily and rapidly detect mutations caused by metals or other stresses.

Requirement of glutathione for Sod1 activation during lifespan extension.

It has been shown that the activation of cytosolic superoxide dismutase (Sod1) in Saccharomyces cerevisiae is only dependent on Ccs1, which is responsible for insertion of copper into the enzyme catalytic center, and that glutathione (GSH) is not necessary for this process. In this work, we addressed an important role of GSH in Sod1 activation by a Ccs1-dependent mechanism during oxidative stress and its role in yeast lifespan. Exponential cells of Saccharomyces cerevisiae, treated or not with 0.5 mM menadione for 1 h, were used for evaluation of the effect of a mild oxidative stress pre-treatment on chronological lifespan. The results showed that menadione induced a lifespan extension in the wild-type (WT) strain but this adaptive response was repressed in gsh1 and in sod1 strains. Interestingly, menadione treatment increased SOD1 and CCS1 gene expression in both WT and gsh1 strains. However, while these strains showed the same Sod1 activity before treatment, only the WT presented an increase of Sod1 activity after menadione exposure. Glutathionylation seems to be essential for Sod1 activation since no increase in activity was observed after menadione treatment in grx1 and grx2 null mutants. Our results suggest that GSH and glutathionylation are fundamental to protect Sod1 sulfhydryl residues under mild oxidative stress, enabling Sod1 activation and lifespan extension.