Amphibian skin is a multifunctional organ that plays key roles in defense, breathing, and water balance. In this study, skin secretion samples of the fire salamander (Salamandra salamandra) were separated using RP-HPLC and de novo sequenced using MALDI-TOF MS/MS. Next, we used an in silico platform to screen antioxidant molecules in the framework of density functional theory. One of the identified peptides, salamandrin-I, [M + H]+ = 1406.6 Da, was selected for solid-phase synthesis; it showed free radical scavenging activity against DPPH and ABTS radicals. Salamandrin-I did not show antimicrobial activity against Gram-positive and -negative bacteria. In vitro assays using human microglia and red blood cells showed that salamandrin-I has no cytotoxicity up to the concentration of 100 µM. In addition, in vivo toxicity tests on Galleria mellonella larvae resulted in no mortality at 20 and 40 mg/kg. Antioxidant peptides derived from natural sources are increasingly attracting interest. Among several applications, these peptides, such as salamandrin-I, can be used as templates in the design of novel antioxidant molecules that may contribute to devising strategies for more effective control of neurological disease.

Background: The need to restructure the world's energy matrix based on fossil fuels and mitigate greenhouse gas emissions stimulated the development of new biobased technologies for renewable energy. One promising and cleaner alternative is the use of second-generation (2G) fuels, produced from lignocellulosic biomass sugars. A major challenge on 2G technologies establishment is the inefficient assimilation of the five-carbon sugar xylose by engineered Saccharomyces cerevisiae strains, increasing fermentation time. The uptake of xylose across the plasma membrane is a critical limiting step and the budding yeast S. cerevisiae is not designed with a broad transport system and regulatory mechanisms to assimilate xylose in a wide range of concentrations present in 2G processes. Results: Assessing diverse microbiomes such as the digestive tract of plague insects and several decayed lignocellulosic biomasses, we isolated several yeast species capable of using xylose. Comparative fermentations selected the yeast Candida sojae as a potential source of high-affinity transporters. Comparative genomic analysis elects four potential xylose transporters whose properties were evaluated in the transporter null EBY.VW4000 strain carrying the xylose-utilizing pathway integrated into the genome. While the traditional xylose transporter Gxf1 allows an improved growth at lower concentrations (10 g/L), strains containing Cs3894 and Cs4130 show opposite responses with superior xylose uptake at higher concentrations (up to 50 g/L). Docking and normal mode analysis of Cs4130 and Gxf1 variants pointed out important residues related to xylose transport, identifying key differences regarding substrate translocation comparing both transporters. Conclusions: Considering that xylose concentrations in second-generation hydrolysates can reach high values in several designed processes, Cs4130 is a promising novel candidate for xylose uptake. Here, we demonstrate a novel eukaryotic molecular transporter protein that improves growth at high xylose concentrations and can be used as a promising target towards engineering efficient pentose utilization in yeast.

Most CRISPR/Cas9 applications in yeast rely on a plasmid-based expression of Cas9 and its guide RNA (gRNA) containing a 20-nucleotides (nts) spacer tailored to each genomic target. The lengthy assembly of this customized gRNA requires at least 3–5 days for its precloning in Escherichia coli, purification, validation, and cotransformation with Cas9 into a yeast strain. Here, we constructed a series of 12 EasyGuide plasmids to simplify CRISPR/Cas9 applications in Saccharomyces cerevisiae. The new vectors provide templates for generating PCR fragments that can assemble up to six functional gRNAs directly into yeasts via homologous recombination between the 20-nts spacers. By dispensing precloning in E. coli, yeast in vivo gRNA assembly significantly reduces the CRISPR/Cas9 experimental workload. A highly efficient yeast genome editing procedure, involving PCR amplification of gRNAs and donors, followed by their transformation into a Cas9-expressing strain, can be easily accomplished through a quick protocol.

The development of high-performance xylosefermenting yeast is essential to achieve feasible conversion of biomass-derived sugars in lignocellulosebased biorefineries. However, engineered C5-strains of Saccharomyces cerevisiae still present low xylose consumption rates under anaerobic conditions. Here, we explore alternative metabolisms involved in metal homeostasis, which positively affect C5 fermentation and analyse the non-obvious regulatory network connection of both metabolisms using time-course transcriptome analysis. Our results indicated the vacuolar Fe 2+ /Mn 2+ transporter CCC1, and the protein involved in heavy metal ion homeostasis BSD2, as promising new targets for rational metabolic engineering strategies, enhancing xylose consumption in nine and 2.3fold compared with control. Notably, intracellular metal concentration levels were affected differently by mutations and the results were compared with positive controls isu1D, a Fe-S cluster scaffold protein, and ssk2D, a component of MAPKKK pathway. Temporal expression profiles indicate a metabolic remodelling in response to xylose, demonstrating changes in the main sugar sensing signalling pathways.