Fighting pathogenic yeasts with plant defensins and anti-fungal proteins from fungi.

Fungal infections represent a significant health risk worldwide. Opportunistic infections caused by yeasts, particularly by Candida spp. and their virulent emerging isolates, have become a major threat to humans, with an increase in fatal cases of infections attributed to the lack of effective anti-yeast therapies and the emergence of fungal resistance to the currently applied drugs. In this regard, the need for novel anti-fungal agents with modes of action different from those currently available is undeniable. Anti-microbial peptides (AMPs) are promising candidates for the development of novel anti-fungal biomolecules to be applied in clinic. A class of AMPs that is of particular interest is the small cysteine-rich proteins (CRPs). Among CRPs, plant defensins and anti-fungal proteins (AFPs) of fungal origin constitute two of the largest and most promising groups of CRPs showing anti-fungal properties, including activity against multi-resistant pathogenic yeasts. In this review, we update and compare the sequence, structure, and properties of plant defensins and AFPs with anti-yeast activity, along with their in vitro and in vivo potency. We focus on the current knowledge about their mechanism of action that may lead the way to new anti-fungals, as well as on the developments for their effective biotechnological production. KEY POINTS: • Plant defensins and fungal AFPs are alternative anti-yeast agents • Their multi-faceted mode of action makes occurrence of resistance rather improbable • Safe and cost-effective biofactories remain crucial for clinical application.

A novel aminotransferase gene and its regulator acquired in Saccharomyces by a horizontal gene transfer event.

BACKGROUND: Horizontal gene transfer (HGT) is an evolutionary mechanism of adaptive importance, which has been deeply studied in wine S. cerevisiae strains, where those acquired genes conferred improved traits related to both transport and metabolism of the nutrients present in the grape must. However, little is known about HGT events that occurred in wild Saccharomyces yeasts and how they determine their phenotypes. RESULTS: Through a comparative genomic approach among Saccharomyces species, we detected a subtelomeric segment present in the S. uvarum, S. kudriavzevii, and S. eubayanus species, belonging to the first species to diverge in the Saccharomyces genus, but absent in the other Saccharomyces species. The segment contains three genes, two of which were characterized, named DGD1 and DGD2. DGD1 encodes dialkylglicine decarboxylase, whose specific substrate is the non-proteinogenic amino acid 2-aminoisobutyric acid (AIB), a rare amino acid present in some antimicrobial peptides of fungal origin. DGD2 encodes putative zinc finger transcription factor, which is essential to induce the AIB-dependent expression of DGD1. Phylogenetic analysis showed that DGD1 and DGD2 are closely related to two adjacent genes present in Zygosaccharomyces. CONCLUSIONS: The presented results show evidence of an early HGT event conferring new traits to the ancestor of the Saccharomyces genus that could be lost in the evolutionary more recent Saccharomyces species, perhaps due to loss of function during the colonization of new habitats.

Novel findings about the mode of action of the antifungal protein PeAfpA against Saccharomyces cerevisiae.

Antifungal proteins (AFPs) from filamentous fungi offer the potential to control fungal infections that threaten human health and food safety. AFPs exhibit broad antifungal spectra against harmful fungi, but limited knowledge of their killing mechanism hinders their potential applicability. PeAfpA from Penicillium expansum shows strong antifungal potency against plant and human fungal pathogens and stands above other AFPs for being active against the yeast Saccharomyces cerevisiae. We took advantage of this and used a model laboratory strain of S. cerevisiae to gain insight into the mode of action of PeAfpA by combining (i) transcriptional profiling, (ii) PeAfpA sensitivity analyses of deletion mutants available in the S. cerevisiae genomic deletion collection and (iii) cell biology studies using confocal microscopy. Results highlighted and confirmed the role of the yeast cell wall (CW) in the interaction with PeAfpA, which can be internalized through both energy-dependent and independent mechanisms. The combined results also suggest an active role of the CW integrity (CWI) pathway and the cAMP-PKA signalling in the PeAfpA killing mechanism. Besides, our studies revealed the involvement of phosphatidylinositol metabolism and the participation of ROX3, which codes for the subunit 19 of the RNA polymerase II mediator complex, in the yeast defence strategy. In conclusion, our study provides clues about both the killing mechanism of PeAfpA and the fungus defence strategies against the protein, suggesting also targets for the development of new antifungals. KEY POINTS: • PeAfpA is a cell-penetrating protein with inhibitory activity against S. cerevisiae. • The CW integrity (CWI) pathway is a key player in the PeAfpA killing mechanism. • Phosphatidylinositol metabolism and ROX3 are involved in the yeast defence strategy.

The Penicillium digitatum antifungal protein PdAfpB shows high activity against mycobiota involved in sliced bread spoilage.

This study aimed to isolate and identify fungal species involved in sliced bread spoilage, and to evaluate their susceptibility to antifungal proteins of fungal origin (AFPs). Proteins include PdAfpB from Penicillium digitatum and PeAfpA, PeAfpB and PeAfpC from Penicillium expansum. Based on morphological criteria, a group of sixteen fungal isolates were selected and subsequently identified at the species level using sequence analysis. Penicillium species, the predominant mycobiota, were identified based on a combined phylogenetic analysis using ITS and ß-tubulin sequences, being P. roqueforti, P. brevicompactum, P. chrysogenum and P. crustosum the most abundant species. Aspergillus versicolor, Aspergillus niger and Bissochlamys spectabilis were also identified. Regarding the antifungal activity of AFPs, PdAfpB and PeAfpA were the most potent proteins since the growth of most of tested fungi was completely inhibited by concentrations ranging from 2 to 32 µg/mL. PeAfpB showed moderate antifungal activity, whereas PeAfpC was the least active protein. The best in vitro AFPs, PdAfpB and PeAfpA, were also evaluated in in situ protection assays against P. roqueforti. PdAfpB provoked a clear reduction of P. roqueforti growth in sliced bread samples, suggesting that this AFP has a protective effect on bread. This study underlines the potential of the AFPs tested, in particular PdAfpB, as alternative antifungal agents for extending sliced bread shelf life.

Application of recyclable CRISPR/Cas9 tools for targeted genome editing in the postharvest pathogenic fungi Penicillium digitatum and Penicillium expansum.

Penicillium digitatum and Penicillium expansum are plant pathogenic fungi that cause the green and blue mold diseases, respectively, leading to serious postharvest economic losses worldwide. Moreover, P. expansum can produce mycotoxins, which are hazardous compounds to human and animal health. The development of tools that allow multiple and precise genetic manipulation of these species is crucial for the functional characterization of their genes. In this sense, CRISPR/Cas9 represents an excellent opportunity for genome editing due to its efficiency, accuracy and versatility. In this study, we developed protoplast generation and transformation protocols and applied them to implement the CRISPR/Cas9 technology in both species for the first time. For this, we used a self-replicative, recyclable AMA1-based plasmid which allows unlimited number of genomic modifications without the limitation of integrative selection markers. As test case, we successfully targeted the wetA gene, which encodes a regulator of conidiophore development. Finally, CRISPR/Cas9-derived ΔwetA strains were analyzed. Mutants showed reduced axenic growth, differential pathogenicity and altered conidiogenesis and germination. Additionally, P. digitatum and P. expansum ΔwetA mutants showed distinct sensitivity to fungal antifungal proteins (AFPs), which are small, cationic, cysteine-rich proteins that have become interesting antifungals to be applied in agriculture, medicine and in the food industry. With this work, we demonstrate the feasibility of the CRISPR/Cas9 system, expanding the repertoire of genetic engineering tools available for these two important postharvest pathogens and open up the possibility to adapt them to other economically relevant phytopathogenic fungi, for which toolkits for genetic modifications are often limited.

Differential susceptibility of mycotoxin-producing fungi to distinct antifungal proteins (AFPs).

The global challenge to prevent fungal spoilage and mycotoxin contamination on foods and feeds require the development of new antifungal strategies. Filamentous fungi encode diverse antifungal proteins (AFPs), which offer a great potential for the control of contaminant fungi. In this study, four AFPs from Penicillium digitatum (PdAfpB) and Penicillium expansum (PeAfpA, PeAfpB and PeAfpC) belonging to classes A, B and C, were tested against a representative panel of mycotoxin-producing fungi. They included a total of 38 strains representing 32 different species belonging to the genera Alternaria, Aspergillus, Byssochlamys, Fusarium and Penicillium. PeAfpA exhibited a potent antifungal activity, since the growth of all tested fungi was completely inhibited by concentrations ranging from 0.5 to 16 µg/mL. PdAfpB and PeAfpB, although less effective than PeAfpA, showed significant activity against most of the mycotoxigenic fungi tested. Importantly, PeAfpC previously described as inactive, showed a powerful inhibition against B. spectabilis strains, which are important spoilage and mycotoxin fungi in pasteurized foods. Although less effective than in liquid media, AFPs affected fungal growth on solid media. This study also underlines the potential of these AFPs, in particular PeAfpA, as future antifungal agents for applications in foods, on growing crops or during postharvest storage.

Antifungal Peptides and Proteins to Control Toxigenic Fungi and Mycotoxin Biosynthesis.

The global challenge to prevent fungal spoilage and mycotoxin contamination on food and feed requires the development of new antifungal strategies. Antimicrobial peptides and proteins (AMPs) with antifungal activity are gaining much interest as natural antifungal compounds due to their properties such as structure diversity and function, antifungal spectrum, mechanism of action, high stability and the availability of biotechnological production methods. Given their multistep mode of action, the development of fungal resistance to AMPs is presumed to be slow or delayed compared to conventional fungicides. Interestingly, AMPs also accomplish important biological functions other than antifungal activity, including anti-mycotoxin biosynthesis activity, which opens novel aspects for their future use in agriculture and food industry to fight mycotoxin contamination. AMPs can reach intracellular targets and exert their activity by mechanisms other than membrane permeabilization. The mechanisms through which AMPs affect mycotoxin production are varied and complex, ranging from oxidative stress to specific inhibition of enzymatic components of mycotoxin biosynthetic pathways. This review presents natural and synthetic antifungal AMPs from different origins which are effective against mycotoxin-producing fungi, and aims at summarizing current knowledge concerning their additional effects on mycotoxin biosynthesis. Antifungal AMPs properties and mechanisms of action are also discussed.

Purification and characterization of an inulinase produced by a Kluyveromyces marxianus strain isolated from blue agave bagasse.

Exo-inulinases are versatile enzymes that have gained attention in recent years due to their ability to hydrolyze linear and branched polyfructose chains found in inulines. Agavin, a branched inulin, is found in Agave plant, the raw matter to produce tequila. Our group has isolated several microbial strains from agave bagasse, an agro-industrial residue from tequila production that increases yearly. Strain ISO3, identified as Kluyveromyces marxianus, showed a remarkable activity towards agavin, and from its fermentation liquor an inulinolytic enzyme (Inu-ISO3) was purified. The isolated enzyme is a glycosylated dimeric protein with a molecular mass of ~256 kDa, as determined by DLS and SEC. The enzyme has an isoelectric pH of 4.6 and has both inulinase and invertase activities with an I/S ratio (ratio of activity with agavin to activity with sucrose) of 1.39. The enzyme has temperature and pH optima of 50 °C and 5.5, respectively, and follows hyperbolic kinetics with agavin (kcat of 339 ± 27 s-1 and KM of 11.8 ± 1.5 mM). The remarkable activity of Inu-ISO3 on linear and branched inulin spotlights this enzyme as a potential player in the treatment of agricultural residua for the generation of added-value products.

Differential roles, crosstalk and response to the Antifungal Protein AfpB in the three Mitogen-Activated Protein Kinases (MAPK) pathways of the citrus postharvest pathogen Penicillium digitatum.

Fungi have three mitogen-activated protein kinases (MAPKs): Kss1/Fus3 involved in the invasive growth and virulence of pathogens, Hog1 in response to osmotic stress, and Slt2/Mpk1 in response to cell wall (CW) stress. We conducted comparative analyses of these MAPKs in the phytopathogen Penicillium digitatum and studied their role in the mode of action of the novel self-antifungal protein AfpB. The sensitivity to different stresses of Δhog1 and the reduced growth of Δkss1 coincided with previous reports. However, Δslt2 showed a strong reduction of growth and conidiation, abnormal morphology, and sensitivity to CW stress and temperature. The complementation of Δslt2 validated this mutant. Immunodetection of P-Hog1 and P-Slt2 confirmed the loss and gain of MAPKs in the mutant and complemented strains. Mutants Δslt2 and Δkss1 showed a strong reduction in virulence, whereas Δhog1 was the least affected, and none sporulated during infection. We studied the MAPK signalling induction in response to different treatments. Our data revealed a complex crosstalk involving the three MAPKs, the differential responses of Hog1 and Slt2 to various stresses and their induction by AfpB or the fungicide fludioxonil (FD). Δhog1 resistance to FD confirmed that Hog1 mediates the activity of FD, whereas Δkss1 sensitivity is probably due to the basal activation of Hog1 in Δkss1. None of the three MAPK mutants showed increased sensitivity to AfpB, contrary to previous reports of other antifungal proteins, which indicates that the observed AfpB-mediated activation of Hog1 and Slt2 would not have a defensive role.

FungalBraid: A GoldenBraid-based modular cloning platform for the assembly and exchange of DNA elements tailored to fungal synthetic biology.

Current challenges in the study and biotechnological exploitation of filamentous fungi are the optimization of DNA cloning and fungal genetic transformation beyond model fungi, the open exchange of ready-to-use and standardized genetic elements among the research community, and the availability of universal synthetic biology tools and rules. The GoldenBraid (GB) cloning framework is a Golden Gate-based DNA cloning system developed for plant synthetic biology through Agrobacterium tumefaciens-mediated genetic transformation (ATMT). In this study, we develop reagents for the adaptation of GB version 3.0 from plants to filamentous fungi through: (i) the expansion of the GB toolbox with the domestication of fungal-specific genetic elements; (ii) the design of fungal-specific GB structures; and (iii) the ATMT and gene disruption of the plant pathogen Penicillium digitatum as a proof of concept. Genetic elements domesticated into the GB entry vector pUPD2 include promoters, positive and negative selection markers and terminators. Interestingly, some GB elements can be directly exchanged between plants and fungi, as demonstrated with the marker hph for HygR or the fluorescent protein reporter YFP. The iterative modular assembly of elements generates an endless number of diverse transcriptional units and other higher order combinations in the pDGB3α/pDGB3Ω destination vectors. Furthermore, the original plant GB syntax was adapted here to incorporate specific GB structures for gene disruption through homologous recombination and dual selection. We therefore have successfully adapted the GB technology for the ATMT of fungi. We propose the name of FungalBraid (FB) for this new branch of the GB technology that provides open, exchangeable and collaborative resources to the fungal research community.

Tryptophan-Containing Dual Neuroprotective Peptides: Prolyl Endopeptidase Inhibition and Caenorhabditis elegans Protection from ß-Amyloid Peptide Toxicity.

Neuroprotective peptides represent an attractive pharmacological strategy for the prevention or treatment of age-related diseases, for which there are currently few effective therapies. Lactoferrin (LF)-derived peptides (PKHs) and a set of six rationally-designed tryptophan (W)-containing heptapeptides (PACEIs) were characterized as prolyl endopeptidase (PEP) inhibitors, and their effect on ß-amyloid peptide (Aß) toxicity in a Caenorhabditis elegans model of Alzheimer's disease (AD) was evaluated. Two LF-derived sequences, PKH8 and PKH11, sharing a W at the C-terminal end, and the six PACEI heptapeptides (PACEI48L to PACEI53L) exhibited significant in vitro PEP inhibition. The inhibitory peptides PKH11 and PACEI50L also alleviated Aß-induced paralysis in the in vivo C. elegans model of AD. Partial or total loss of the inhibitory effect on PEP was achieved by the substitution of W residues in PKH11 and PACEI50L and correlated with the loss of protection against Aß toxicity, pointing out the relevance of W on the neuroprotective activity. Further experiments suggest that C. elegans protection might not be mediated by an antioxidant mechanism but rather by inhibition of Aß oligomerization and thus, amyloid deposition. In conclusion, novel natural and rationally-designed W-containing peptides are suitable starting leads to design effective neuroprotective agents.

A Penicillium chrysogenum-based expression system for the production of small, cysteine-rich antifungal proteins for structural and functional analyses.

BACKGROUND: Small, cysteine-rich and cationic antifungal proteins (APs) from filamentous ascomycetes, such as NFAP from Neosartorya fischeri and PAF from Penicillium chrysogenum, are promising candidates for novel drug development. A prerequisite for their application is a detailed knowledge about their structure-function relation and mode of action, which would allow protein modelling to enhance their toxicity and specificity. Technologies for structure analyses, such as electronic circular dichroism (ECD) or NMR spectroscopy, require highly purified samples and in case of NMR milligrams of uniformly 15N-/13C-isotope labelled protein. To meet these requirements, we developed a P. chrysogenum-based expression system that ensures sufficient amount and optimal purity of APs for structural and functional analyses. RESULTS: The APs PAF, PAF mutants and NFAP were expressed in a P. chrysogenum ∆paf mutant strain that served as perfect microbial expression factory. This strain lacks the paf-gene coding for the endogenous antifungal PAF and is resistant towards several APs from other ascomycetes. The expression of the recombinant proteins was under the regulation of the strong paf promoter, and the presence of a paf-specific pre-pro sequence warranted the secretion of processed proteins into the supernatant. The use of defined minimal medium allowed a single-step purification of the recombinant proteins. The expression system could be extended to express PAF in the related fungus Penicillium digitatum, which does not produce detectable amounts of APs, demonstrating the versatility of the approach. The molecular masses, folded structures and antifungal activity of the recombinant proteins were analysed by ESI-MS, ECD and NMR spectroscopy and growth inhibition assays. CONCLUSION: This study demonstrates the implementation of a paf promoter driven expression cassettes for the production of cysteine-rich, cationic, APs in different Penicillium species. The system is a perfect tool for the generation of correctly folded proteins with high quality for structure-function analyses.

Concatemerization increases the inhibitory activity of short, cell-penetrating, cationic and tryptophan-rich antifungal peptides.

There are short cationic and tryptophan-rich antifungal peptides such as the hexapeptide PAF26 (RKKWFW) that have selective toxicity and cell penetration properties against fungal cells. This study demonstrates that concatemeric peptides with tandem repeats of the heptapeptide PAF54 (which is an elongated PAF26 sequence) show increased fungistatic and bacteriostatic activities while maintaining the absence of hemolytic activity of the monomer. The increase in antimicrobial activity of the double-repeated PAF sequences (diPAFs), compared to the nonrepeated PAF, was higher (4-8-fold) than that seen for the triple-repeated sequences (triPAFs) versus the diPAFs (2-fold). However, concatemerization diminished the fungicidal activity against quiescent spores of the filamentous fungus Penicillium digitatum. Peptide solubility and sensitivity to proteolytic degradation were affected by the design of the concatemers: incorporation of the AGPA sequence hinge to separate PAF54 repeats increased solubility while the C-terminal addition of the KDEL sequence decreased in vitro stability. These results led to the design of the triPAF sequence PAF102 of 30 amino acid residues, with increased antimicrobial activity and minimal inhibitory concentration (MIC) value of 1-5 µM depending on the fungus. Further characterization of the mode-of-action of PAF102 demonstrated that it colocalizes first with the fungal cell wall, it is thereafter internalized in an energy dependent manner into hyphal cells of the filamentous fungus Fusarium proliferatum, and finally kills hyphal cells intracellularly. Therefore, PAF102 showed mechanistic properties against fungi similar to the parental PAF26. These observations are of high interest in the future development of PAF-based antimicrobial molecules optimized for their production in biofactories.

Production of lactulose oligosaccharides by isomerisation of transgalactosylated cheese whey permeate obtained by ß-galactosidases from dairy Kluyveromyces.

ß-Galactosidases from Kluyveromyces lactis and Kluyveromyces marxianus isolated from artisanal ewes' milk cheeses, were used to transgalactosylate lactose from cheese whey permeate (WP). The content of galactooligosaccharides (GOS) obtained by transgalactosylation was comparable with that formed using pure lactose as substrate. In order to obtain a mixture with higher prebiotic oligosaccharide content, isomerisation of the transgalactosylated WP was carried out using sodium aluminate as catalyst. The transgalactosylated mixtures at 6 h of reaction contained amounts of prebiotic carbohydrates (tagatose, lactulose, GOS and oligosaccharides derived from lactulose, OsLu) close to 50 g/100 g of total carbohydrates for all the strains tested, corresponding to 322 g prebiotics/kg whey permeate. Thus, the suitability of this methodology to produce mixtures of dietary non-digestible carbohydrates with prebiotic properties from WP has been demonstrated, which is interesting for the food industry since it increases the value and the applicability of this by-product from cheese manufacture.

Yeast species and genetic heterogeneity within Debaryomyces hansenii along the ripening process of traditional ewes' and goats' cheeses.

The yeasts present during the ripening process of ewes' and goats' cheeses produced in a small traditional dairy in Mediterranean Spain were isolated and identified. Five hundred and thirty strains pertaining to eleven yeast species representing eight genera were identified using molecular methods. Debaryomyces hansenii was the yeast species most frequently isolated in all cheeses. Other yeast species commonly found in dairy products were present at the first maturing weeks. Two yeast species Trichosporon coremiiforme and Trichosporon domesticum have been reported in cheeses for the first time, and Meyerozyma guilliermondii has been newly isolated from goats' cheeses. Yeast species composition changed greatly along the process; although, D. hansenii dominated at the end of ripening in all cheeses. Most yeast isolates were able to hydrolyze casein and fatty acid esters. One hundred and eighty seven D. hansenii isolates were genotyped by PCR amplification of M13 satellites and an UPGMA dendrogram was constructed. The majority of isolates were grouped in 5 clusters while 7 profiles were represented by 1-3 isolates. These results demonstrate the genetic heterogeneity present in D. hansenii strains isolated from raw milk cheeses.

Increasing the efficiency of CRISPR/Cas9-mediated genome editing in the citrus postharvest pathogen Penicillium digitatum.

BACKGROUND: Penicillium digitatum is a fungal plant pathogen that causes the green mold disease in harvested citrus fruits. Due to its economical relevance, many efforts have focused on the development of genetic engineering tools for this fungus. Adaptation of the CRISPR/Cas9 technology was previously accomplished with self-replicative AMA1-based plasmids for marker-free gene editing, but the resulting efficiency (10%) limited its practical implementation. In this study, we aimed to enhance the efficiency of the CRISPR/Cas9-mediated gene editing in P. digitatum to facilitate its practical use. RESULTS: Increasing the culture time by performing additional culture streaks under selection conditions in a medium that promotes slower growth rates significantly improved the gene editing efficiency in P. digitatum up to 54-83%. To prove this, we disrupted five candidate genes that were chosen based on our previous high-throughput gene expression studies aimed at elucidating the transcriptomic response of P. digitatum to the antifungal protein PdAfpB. Two of these genes lead to visual phenotypic changes (PDIG_53730/pksP, and PDIG_54100/arp2) and allowed to start the protocol optimization. The other three candidates (PDIG_56860, PDIG_33760/rodA and PDIG_68680/dfg5) had no visually associated phenotype and were targeted to confirm the high efficiency of the protocol. CONCLUSION: Genome editing efficiency of P. digitatum was significantly increased from 10% to up to 83% through the modification of the selection methodology, which demonstrates the feasibility of the CRISPR/Cas9 system for gene disruption in this phytopathogenic fungus. Moreover, the approach described in this study might help increase CRISPR/Cas9 gene editing efficiencies in other economically relevant fungal species for which editing efficiency via CRISPR/Cas9 is still low.

Studies on the biological role of the antifungal protein PeAfpA from Penicillium expansum by functional gene characterization and transcriptomic profiling.

Antifungal proteins (AFPs) from filamentous fungi have enormous potential as novel biomolecules for the control of fungal diseases. However, little is known about the biological roles of AFPs beyond their antifungal action. Penicillium expansum encodes three phylogenetically different AFPs (PeAfpA, PeAfpB and PeAfpC) with diverse profiles of antifungal activity. PeAfpA stands out as a highly active AFP that is naturally produced at high yields. Here, we provide new data about the function of PeAfpA in P. expansum through phenotypical characterization and transcriptomic studies of null mutants of the corresponding afpA gene. Mutation of afpA did not affect axenic growth, conidiation, virulence, stress responses or sensitivity towards P. expansum AFPs. However, RNA sequencing evidenced a massive transcriptomic change linked to the onset of PeAfpA production. We identified two large gene expression clusters putatively involved in PeAfpA function, which correspond to genes induced or repressed with the production of PeAfpA. Functional enrichment analysis unveiled significant changes in genes related to fungal cell wall remodeling, mobilization of carbohydrates and plasma membrane transporters. This study also shows a putative co-regulation between the three afp genes. Overall, our transcriptomic analyses provide valuable insights for further understanding the biological functions of AFPs.

Bioethanol lignin-rich residue from olive stones for electrospun nanostructures development and castor oil structuring.

This work describes the chemical and structural characterization of a lignin-rich residue from the bioethanol production of olive stones and its use for nanostructures development by electrospinning and castor oil structuring. The olive stones were treated by sequential acid/steam explosion pretreatment, further pre-saccharification using a hydrolytic enzyme, and simultaneous saccharification and fermentation (PSSF). The chemical composition of olive stone lignin-rich residue (OSL) was evaluated by standard analytical methods, showing a high lignin content (81.3 %). Moreover, the structural properties were determined by Fourier-transform infrared spectroscopy, nuclear magnetic resonance, and size exclusion chromatography. OSL showed a predominance of ß-ß' resinol, followed by ß-O-4' alkyl aryl ethers and ß-5' phenylcoumaran substructures, high molecular weight, and low S/G ratio. Subsequently, electrospun nanostructures were obtained from solutions containing 20 wt% OSL and cellulose triacetate with variable weight ratios in N, N-Dimethylformamide/Acetone blends and characterized by scanning electron microscopy. Their morphologies were highly dependent on the rheological properties of polymeric solutions. Gel-like dispersions can be obtained by dispersing the electrospun OSL/CT bead nanofibers and uniform nanofiber mats in castor oil. The rheological properties were influenced by the membrane concentration and the OSL:CT weight ratio, as well as the morphology of the electrospun nanostructures.

Recent advances on physical technologies for the pretreatment of food waste and lignocellulosic residues.

The complete deployment of a bio-based economy is essential to meet the United Nations' Sustainable Development Goals from the 2030 Agenda. In this context, food waste and lignocellulosic residues are considered low-cost feedstocks for obtaining industrially attractive products through biological processes. The effective conversion of these raw materials is, however, still challenging, since they are recalcitrant to bioprocessing and must be first treated to alter their physicochemical properties and ease the accessibility to their structural components. Among the full pallet of pretreatments, physical methods are recognised to have a high potential to transform food waste and lignocellulosic residues. This review provides a critical discussion about the recent advances on milling, extrusion, ultrasound, and microwave pretreatments. Their mechanisms and modes of application are analysed and the main drawbacks and limitations for their use at an industrial scale are discussed.

Rationally designed antifungal protein chimeras reveal new insights into structure-activity relationship.

Antifungal proteins (AFPs) are promising antimicrobial compounds that represent a feasible alternative to fungicides. Penicillium expansum encodes three phylogenetically distinct AFPs (PeAfpA, PeAfpB and PeAfpC) which show different antifungal profiles and fruit protection effects. To gain knowledge about the structural determinants governing their activity, we solved the crystal structure of PeAfpB and rationally designed five PeAfpA::PeAfpB chimeras (chPeAFPV1-V5). Chimeras showed significant differences in their antifungal activity. chPeAFPV1 and chPeAFPV2 improved the parental PeAfpB potency, and it was very similar to that of PeAfpA. chPeAFPV4 and chPeAFPV5 showed an intermediate profile of activity compared to the parental proteins while chPeAFPV3 was inactive towards most of the fungi tested. Structural analysis of the chimeras evidenced an identical scaffold to PeAfpB, suggesting that the differences in activity are due to the contributions of specific residues and not to induced conformational changes or structural rearrangements. Results suggest that mannoproteins determine protein interaction with the cell wall and its antifungal activity while there is not a direct correlation between binding to membrane phospholipids and activity. This work provides new insights about the relevance of sequence motifs and the feasibility of modifying protein specificity, opening the door to the rational design of chimeras with biotechnological applicability.