Antimicrobial Properties of New Polyamines Conjugated with Oxygen-Containing Aromatic Functional Groups.

Antibiotic resistance is now a first-order health problem, which makes the development of new families of antimicrobials imperative. These compounds should ideally be inexpensive, readily available, highly active, and non-toxic. Here, we present the results of our investigation regarding the antimicrobial activity of a series of natural and synthetic polyamines with different architectures (linear, tripodal, and macrocyclic) and their derivatives with the oxygen-containing aromatic functional groups 1,3-benzodioxol, ortho/para phenol, or 2,3-dihydrobenzofuran. The new compounds were prepared through an inexpensive process, and their activity was tested against selected strains of yeast, as well as Gram-positive and Gram-negative bacteria. In all cases, the conjugated derivatives showed antimicrobial activity higher than the unsubstituted polyamines. Several factors, such as the overall charge at physiological pH, lipophilicity, and the topology of the polyamine scaffold were relevant to their activity. The nature of the lipophilic moiety was also a determinant of human cell toxicity. The lead compounds were found to be bactericidal and fungistatic, and they were synergic with the commercial antifungals fluconazole, cycloheximide, and amphotericin B against the yeast strains tested.

Biotechnological applications of biofilms formed by osmotolerant and halotolerant yeasts.

Many microorganisms are capable of developing biofilms under adverse conditions usually related to nutrient limitation. They are complex structures in which cells (in many cases of different species) are embedded in the material that they secrete, the extracellular matrix (ECM), which is composed of proteins, carbohydrates, lipids, and nucleic acids. The ECM has several functions including adhesion, cellular communication, nutrient distribution, and increased community resistance, this being the main drawback when these microorganisms are pathogenic. However, these structures have also proven useful in many biotechnological applications. Until now, the most interest shown in these regards has focused on bacterial biofilms, and the literature describing yeast biofilms is scarce, except for pathological strains. Oceans and other saline reservoirs are full of microorganisms adapted to extreme conditions, and the discovery and knowledge of their properties can be very interesting to explore new uses. Halotolerant and osmotolerant biofilm-forming yeasts have been employed for many years in the food and wine industry, with very few applications in other areas. The experience gained in bioremediation, food production and biocatalysis with bacterial biofilms can be inspiring to find new uses for halotolerant yeast biofilms. In this review, we focus on the biofilms formed by halotolerant and osmotolerant yeasts such as those belonging to Candida, Saccharomyces flor yeasts, Schwannyomyces or Debaryomyces, and their actual or potential biotechnological applications. KEY POINTS: • Biofilm formation by halotolerant and osmotolerant yeasts is reviewed. • Yeasts biofilms have been widely used in food and wine production. • The use of bacterial biofilms in bioremediation can be expanded to halotolerant yeast counterparts.

Recent developments in the biology and biotechnological applications of halotolerant yeasts.

Natural hypersaline environments are inhabited by an abundance of prokaryotic and eukaryotic microorganisms capable of thriving under extreme saline conditions. Yeasts represent a substantial fraction of halotolerant eukaryotic microbiomes and are frequently isolated as food contaminants and from solar salterns. During the last years, a handful of new species has been discovered in moderate saline environments, including estuarine and deep-sea waters. Although Saccharomyces cerevisiae is considered the primary osmoadaptation model system for studies of hyperosmotic stress conditions, our increasing understanding of the physiology and molecular biology of halotolerant yeasts provides new insights into their distinct metabolic traits and provides novel and innovative opportunities for genome mining of biotechnologically relevant genes. Yeast species such as Debaryomyces hansenii, Zygosaccharomyces rouxii, Hortaea werneckii and Wallemia ichthyophaga show unique properties, which make them attractive for biotechnological applications. Select halotolerant yeasts are used in food processing and contribute to aromas and taste, while certain gene clusters are used in second generation biofuel production. Finally, both pharmaceutical and chemical industries benefit from applications of halotolerant yeasts as biocatalysts. This comprehensive review summarizes the most recent findings related to the biology of industrially-important halotolerant yeasts and provides a detailed and up-to-date description of modern halotolerant yeast-based biotechnological applications.

Formation and characterization of biofilms formed by salt-tolerant yeast strains in seawater-based growth medium.

Yeast whole cells have been widely used in modern biotechnology as biocatalysts to generate numerous compounds of industrial, chemical, and pharmaceutical importance. Since many of the biocatalysis-utilizing manufactures have become more concerned about environmental issues, seawater is now considered a sustainable alternative to freshwater for biocatalytic processes. This approach plausibly commenced new research initiatives into exploration of salt-tolerant yeast strains. Recently, there has also been a growing interest in possible applications of microbial biofilms in the field of biocatalysis. In these complex communities, cells demonstrate higher resistance to adverse environmental conditions due to their embedment in an extracellular matrix, in which physical, chemical, and physiological gradients exist. Considering these two topics, seawater and biofilms, in this work, we characterized biofilm formation in seawater-based growth media by several salt-tolerant yeast strains with previously demonstrated biocatalytic capacities. The tested strains formed both air-liquid-like biofilms and biofilms on silicone surfaces, with Debaryomyces fabryi, Schwanniomyces etchellsii, Schwanniomyces polymorphus, and Kluyveromyces marxianus showing the highest biofilm formation. The extracted biofilm extracellular matrices mostly consisted of carbohydrates and proteins. The latter group was primarily represented by enzymes involved in metabolic processes, particularly the biosynthetic ones, and in the response to stimuli. Specific features were also found in the carbohydrate composition of the extracellular matrix, which were dependent both on the yeast isolate and the nature of formed biofilms. Overall, our findings presented herein provide a unique data resource for further development and optimization of biocatalytic processes and applications employing seawater and halotolerant yeast biofilms.Key points• Ability for biofilm formation of some yeast-halotolerant strains in seawater medium• ECM composition dependent on strain and biofilm-forming surface• Metabolic enzymes in the ECM with potential applications for biocatalysis.

Surface display of HFBI and DewA hydrophobins on Saccharomyces cerevisiae modifies tolerance to several adverse conditions and biocatalytic performance.

Hydrophobins are relatively small proteins produced naturally by filamentous fungi with interesting biotechnological and biomedical applications given their self-assembly capacity, efficient adherence to natural and artificial surfaces, and to introduce modifications on the hydrophobicity/hydrophilicity of surfaces. In this work we demonstrate the efficient expression on the S. cerevisiae cell surface of class II HFBI of Trichoderma reesei and class I DewA of Aspergillus nidulans, a hydrophobin not previously exposed, using the Yeast Surface Display a-agglutinin (Aga1-Aga2) system. We show that the resulting modifications affect surface properties, and also yeast cells' resistance to several adverse conditions. The fact that viability of the engineered strains increases under heat and osmotic stress is particularly interesting. Besides, improved biocatalytic activity toward the reduction of ketone 1-phenoxypropan-2-one takes place in the reactions carried out at both 30 °C and 40 °C, within a concentration range between 0.65 and 2.5 mg/mL. These results suggest interesting potential applications for hydrophobin-exposing yeasts. KEY POINTS : • Class I hydrophobin DewA can be efficiently exposed on S. cerevisiae cell surfaces. • Yeast exposure of HFBI and DewA increases osmotic and heat resistance. • Engineered strains show modified biocatalytic behavior.

Whole-Cell Biocatalysis in Seawater: New Halotolerant Yeast Strains for the Regio- and Stereoselectivity Reduction of 1-Phenylpropane-1,2-Dione in Saline-Rich Media.

The application of green chemistry concepts in catalysis has considerably increased in recent years, and the interest in using sustainable solvents in the chemical industry is growing. One of the recent proposals to fall in line with this is to employ seawater as a solvent in biocatalytic processes. This involves selecting halotolerant strains capable of carrying out chemical conversions in the presence of the salt concentrations found in this solution. Recent studies by our group have revealed the interest in using strains belonging to Debaryomyces and Schwanniomyces for catalytic processes run in this medium. In the present work, we select other yeasts based on their halotolerance to widen the scope of this strategy. We consider them for the monoreduction of 1-phenylpropane-1,2-dione, a well-characterized reaction that produces acyloin intermediates of pharmaceutical interest. The results obtained herein indicate that using seawater as a solvent for this reaction is possible. The best ones were obtained for Saccharomyces cerevisiae FY86 and Kluyveromyces marxianus, for which acyloins with different stereochemistry were obtained with good to excellent enantiomeric excess.

Biotransformation using halotolerant yeast in seawater: a sustainable strategy to produce R-(-)-phenylacetylcarbinol.

Acyloin condensation between benzaldehyde and decarboxylated pyruvate results in the production of R-(-)-phenylacetylcarbinol, a chiral precursor of the drug ephedrine. Huge research efforts have been made to improve the conditions of this reaction and to avoid the generation of by-products. Recently, we reported the advantages of using whole cells of the yeast Debaryomyces etchellsii as biocatalysts for this purpose. In this work, a new strategy, which fulfills green chemistry principles, is proposed and is based on using seawater as a gentle solvent. We demonstrate that, under these conditions, several improvements can be made compared to employing freshwater: (1) the conversion of the starting material in (R)-PAC is higher and with a minimum production of by-products; (2) it is possible to increase at least twofold the benzaldehyde load in the reaction medium; (3) cells can maintain their activity after several recycling rounds, which makes (R)-PAC production an easy and economical process.

Yeast arming systems: pros and cons of different protein anchors and other elements required for display.

Yeast display is a powerful strategy that consists in exposing peptides or proteins of interest on the cell surface of this microorganism. Ever since initial experiments with this methodology were carried out, its scope has extended and many applications have been successfully developed in different science and technology fields. Several yeast display systems have been designed, which all involve introducting into yeast cells the gene fusions that contain the coding regions of a signal peptide, an anchor protein, to properly attach the target to the cell surface, and the protein of interest to be exposed, all of which are controlled by a strong promoter. In this work, we report the description of such elements for the alternative systems introduced by focusing particularly on anchor proteins. The comparisons made between them are included whenever possible, and the main advantages and inconveniences of each one are discussed. Despite the huge number of publications on yeast surface display and the revisions published to date, this topic has not yet been widely considered. Finally, given the growing interest in developing systems for non-Saccharomyces yeasts, the main strategies reported for some are also summarized.

Development of a new yeast surface display system based on Spi1 as an anchor protein.

Yeast surface display is a powerful tool widely used for many biotechnological and biomedical applications. It consists in exposing peptides and proteins of interest on the surface of Saccharomyces cerevisiae and other yeasts. These molecules are fused to the amino or carboxy terminus of an appropriate cell wall protein, usually bound by glycosylphosphatidylinositol. Several systems for this purpose have been reported to date. In this work, we describe a new yeast surface display strategy based on cell wall protein Spi1 as an anchor, which is expressed in centromeric and episomal plasmids under the control of its own promoter or that corresponding to the PGK1 glycolytic gene. Exposure efficiency was demonstrated by western blot, flow cytometry analyses, and fluorescence microscopy by taking advantage of including the V5 epitope. We also demonstrated the ability of this new strategy for the exposure of several peptides and proteins of different sizes. The regulation of the SPI1 promoter by the stationary phase and other stress conditions reveals interesting potential applications of systems based on it for industrial and biotechnological processes.

Biocatalytic reduction of racemic 2-arenoxycycloalkanones by yeasts P. glucozyma and C. glabrata: one way of achieving chiral 2-arenoxycycloalcohols.

Chiral ß-aryloxy alcohols are interesting building blocks that form part of drugs like ß adrenergic antagonists. Acquiring cyclic rigid analogs to obtain more selective drugs is interesting. Thus, we used whole cells of yeast strains Pichia glucozyma and Candida glabrata to catalyze the reduction of several 2-arenoxycycloalkanones to produce chiral 2-arenoxycycloalcohols with good/excellent enantioselectivity. In both cases, the alcohol configuration that resulted from the carbonyl group reduction was S. Yeast P. glucozyma allowed the conversion of both enantiomers of the starting material to produce 2-arenoxycycloalcohols with configuration (1S, 2R) and (1S, 2S). The reaction with C. glabrata nearly always allowed the kinetic resolution of the starting ketone, recovering 2-arenoxycycloalkanone with configuration S and (1S, 2R)-2-arenoxycycloalcohol.All the four possible stereoisomers of 2-phenoxycyclohexanol and the two enantiomers of 2-phenoxycyclohexanone were obtained by combining the biocatalyzed reaction with the oxidation/reduction of the chiral compounds with standard reagents. This is a simple approach for the synthesis of the rigid chiral moiety 2-arenoxycycloalcohols contained in putative ß-blockers 2-arenoxycycloalkanepropanolamines.

Potential of some yeast strains in the stereoselective synthesis of (R)-(-)-phenylacetylcarbinol and (S)-(+)-phenylacetylcarbinol and their reduced 1,2-dialcohol derivatives.

Whole cells of different yeast species have been widely used for a number of asymmetric transformations. In the present study, the screening of several yeast strains revealed the utility of Debaryomyces etchellsii in acyloin condensation for (R)-(-)-phenylacetylcarbinol production. Some conditions for the efficient biotransformation of benzaldehyde and minimization in the production of by-products were explored: pH of the reaction medium, use of additives (ethanol or acetonitrile), temperature, time, and substrate concentration and dosing. The optimal conditions found allowed the transformation of up to 10 g/L of the starting material in reactions carried out at high scale. Furthermore, the yeast Kluyveromyces marxianus was seen to be a convenient biocatalyst to carry out the kinetic resolution by the bioreduction of racemic (+/-)-phenylacetylcarbinol, resulting in (S)-(+)-phenylacetylcarbinol with excellent stereoselectivity. Finally, the ketone reduction of both isolated stereoisomers (R and S) by D. etchellsii allowed the obtainment of two of the four diastereoisomers of 1-phenyl-1,2-propanediol. All these compounds are key precursors for the production of interesting pharmaceutical and chemical products.

Antimicrobial peptides and their superior fluorinated analogues: structure-activity relationships as revealed by NMR spectroscopy and MD calculations.

The conformations of two synthetic pentapeptides with antimicrobial activity and their 4-fluorophenylalanine (Pff)-containing analogues (ArXArXAr-NH(2); Ar=Phe, Pff; X=Lys, Arg) have been studied. NMR experiments carried out both in aqueous fluoroalcohol solutions and SDS micelles permitted their interactions with membrane-like environments to be explored. WaterLOGSY experiments and Mn(2+)-based paramagnetic probes were also applied to assess their orientations with respect to the SDS micelles. In addition, pulse-field gradient (PFG) diffusion NMR spectroscopy studies were conducted, under different experimental conditions (i.e., concentration, temperature) to characterize the possible changes in the peptides' aggregation states as a putative critical factor for their antimicrobial activity. Finally, molecular dynamics simulations on a variety of conformations showed the intrinsic flexibility of these peptides in both aqueous solutions and membrane-mimetic systems.

The introduction of fluorine atoms or trifluoromethyl groups in short cationic peptides enhances their antimicrobial activity.

The effect of introducing fluorine atoms or trifluoromethyl groups in either the peptidic chain or the C-terminal end of cationic pentapeptides is reported. Three series of amide and ester peptides were synthesised and their antimicrobial properties evaluated. An enhanced activity was found in those derivatives whose structure contained fluorine, suggesting an increase in their hydrophobicity.

On the importance of carbohydrate-aromatic interactions for the molecular recognition of oligosaccharides by proteins: NMR studies of the structure and binding affinity of AcAMP2-like peptides with non-natural naphthyl and fluoroaromatic residues.

The specific interaction of a variety of modified hevein domains to chitooligosaccharides has been studied by NMR spectroscopy in order to assess the importance of aromatic-carbohydrate interactions for the molecular recognition of neutral sugars. These mutant AcAMP2-like peptides, which have 4-fluoro-phenylalanine, tryptophan, or 2-naphthylalanine at the key interacting positions, have been prepared by solid-phase synthesis. Their three-dimensional structures, when bound to the chitin-derived trisaccharide, have been deduced by NMR spectroscopy. By using DYANA and restrained molecular dynamics simulations with the AMBER 5.0 force field, the three-dimensional structures of the protein-sugar complexes have been obtained. The thermodynamic analysis of the interactions that occur upon complex formation have also been carried out. Regarding binding affinity, the obtained data have permitted the deduction that the larger the aromatic group, the higher the association constant and the binding enthalpy. In all cases, entropy opposes binding. In contrast, deactivation of the aromatic rings by attaching fluorine atoms decreases the binding affinity, with a concomitant decrease in enthalpy. The role of the chemical nature of the aromatic ring for establishing sugar contacts has been thus evaluated.

H-Bonding Interactions in the Epoxidation of Alkenylammonium Salts with Dimethyldioxirane and m-Chloroperbenzoic Acid: A Kinetic Study.

The epoxidation rate constants for the reaction of allylic and homoallylic primary and quaternary ammonium salts with DMDO (1b) and m-CPBA (2), as well as the stereochemical outcome of these reactions, were determined. The presence of an ionic functional group in the substrate complicates the kinetic study of the reaction. However, k(0) can be determined from the k(obs) values measured in solutions with different ionic strengths. The order of magnitude of the rate constants is the same for the epoxidation of primary and quaternary homoallylic ammonium salts, while primary allylic ammonium salts react more than 10 times faster than their quaternary counterparts. High syn-diastereoselectivity is achieved in the epoxidation of the primary allylic salt 3aH(+)() while the quaternary allylic ammonium salt 5a(+)() gives equimolecular (m-CPBA) or predominantly anti(DMDO) mixtures of diastereomers. These results are consistent with the existence of hydrogen bond interaction between the protic substrates and the oxidant.