Publications

Sugar beet pulp (SBP), sugar beet molasses (SBM) and unfermented grape marcs (UGM) represent important waste in the agro-food sector. If suitably pre-treated, hexose and pentose sugars can be released in high quantities and can subsequently be used by appropriate cell factories as growth media and for the production of (complex) biomolecules, accomplishing the growing demand for products obtained from sustainable resources. One example is vitamin B9 or folate, a B-complex vitamin currently produced by chemical synthesis, almost exclusively in the oxidized form of folic acid (FA). It is therefore desirable to develop novel competitive strategies for replacing its current fossil-based production with a sustainable bio-based process. In this study, we assessed the production of natural folate by the yeast Scheffersomyces stipitis, investigating SBM, SBP and UGM as potential growth media. Pre-treatment of SBM and SBP had previously been optimized in our laboratory; thus, here we focused only on UGM pre-treatment and hydrolysis strategies for the release of fermentable sugars. Then, we optimized the growth of S. stipitis on the three media formulated from those biomasses, working on inoculum pre-adaptation, oxygen availability and supplementation of necessary nutrients to support the microorganism. Folate production, measured with a microbiological assay, reached 188.2 ± 24.86 μg/L on SBM, 130.6 ± 1.34 μg/L on SBP and 101.9 ± 6.62 μg/L on UGM. Here, we demonstrate the flexibility of S. stipitis in utilizing different residual biomasses as growth media. Moreover, we assessed the production of folate from waste, and to the best of our knowledge, we obtained the highest production of folate from residual biomasses ever reported, providing the first indications for the future development of this microbial production process.

The strong antifungal effect of sulfonated polystyrene nanoparticles (NPs) with an encapsulated tetraphenylporphyrin (TPP) photosensitizer is reported here. TPP is activated by visible light, resulting in the generation of singlet oxygen. Its antifungal action is potentiated in the presence of potassium iodide, yielding I2/I3⁻, another antifungal species. The NPs exhibit no dark toxicity, but a broad spectrum of antifungal photodynamic effects. The efficiency of this rapid killing (on the order of minutes) depends on the concentration of TPP NPs, potassium iodide, yeast species and temperature. A strong antifungal activity of TPP NPs is demonstrated on eleven pathogenic and opportunistic pathogenic yeast species (six Candida species and other yeast species, including melanized Hortaea werneckii). The composition and architecture of yeast cell envelope structures clearly influence the efficacy of photodynamic therapy. Candida krusei is the most sensitive to photodynamic therapy. Despite expectations, melanin does not provide Hortaea cells with marked resistance compared to white yeast species. The kinetics of the interaction of NPs with yeast cells is also described. This study may inspire and promote the fabrication of a new type of antiseptic for various skin injuries in clinical medicine.

In the model yeast Saccharomyces cerevisiae, Trk1 is the main K+ importer. It is involved in many important physiological processes, such as the maintenance of ion homeostasis, cell volume, intracellular pH, and plasma-membrane potential. The ScTrk1 protein can be of great interest to industry, as it was shown that changes in its activity influence ethanol production and tolerance in S. cerevisiae and also cell performance in the presence of organic acids or high ammonium under low K+ conditions. Nonconventional yeast species are attracting attention due to their unique properties and as a potential source of genes that encode proteins with unusual characteristics. In this work, we aimed to study and compare Trk proteins from Debaryomyces hansenii, Hortaea werneckii, Kluyveromyces marxianus, and Yarrowia lipolytica, four biotechnologically relevant yeasts that tolerate various extreme environments. Heterologous expression in S. cerevisiae cells lacking the endogenous Trk importers revealed differences in the studied Trk proteins’ abilities to support the growth of cells under various cultivation conditions such as low K+ or the presence of toxic cations, to reduce plasma-membrane potential or to take up Rb+. Examination of the potential of Trks to support the stress resistance of S. cerevisiae wild-type strains showed that Y. lipolytica Trk1 is a promising tool for improving cell tolerance to both low K+ and high salt and that the overproduction of S. cerevisiae’s own Trk1 was the most efficient at improving the growth of cells in the presence of highly toxic Li+ ions.

The dairy industry processes vast amounts of milk and generates high amounts of secondary by-products, which are still rich in nutrients (high Chemical Oxygen Demand (COD) and Biochemical Oxygen Demand (BOD) levels) but contain high concentrations of salt. The current European legislation only allows disposing of these effluents directly into the waterways with previous treatment, which is laborious and expensive. Therefore, as much as possible, these by-products are reutilized as animal feed material and, if not applicable, used as fertilizers adding phosphorus, potassium, nitrogen, and other nutrients to the soil. Finding biological alternatives to revalue dairy by-products is of crucial interest in order to improve the utilization of dry dairy matter and reduce the environmental impact of every litre of milk produced. Debaryomyces hansenii is a halotolerant non-conventional yeast with high potential for this purpose. It presents some beneficial traits – capacity to metabolize a variety of sugars, tolerance to high osmotic environments, resistance to extreme temperatures and pHs – that make this yeast a well-suited option to grow using complex feedstock, such as industrial waste, instead of the traditional commercial media. In this work, we study for the first time D. hansenii’s ability to grow and produce a recombinant protein (YFP) from dairy saline whey by-products. Cultivations at different scales (1.5, 100 and 500 ml) were performed without neither sterilizing the medium nor using pure water. Our results conclude that D. hansenii is able to perform well and produce YFP in the aforementioned salty substrate. Interestingly, it is able to outcompete other microorganisms present in the waste without altering its cell performance or protein production capacity.

Yeast mannoproteins are proposed as a paraprobiotics with antimicrobial and prebiotic properties. They can be used as biopreservatives in food and in diseases therapies. The knowledge about the specificity and/or capability of their influence on the growth of different microorganism is limited. The study determined the effect of mannoprotein preparations of Saccharomyces cerevisiae (S. cerevisiae) ATCC 7090 and nonconventional yeast origin [Metschnikowia reukaufii (M. reukaufii) WLP 4650 and Wickerhamomyces anomalus (W. anomalus) CCY 38-1-13] on the growth of selected bacteria of the genera: Lactobacilllus, Limosilatobacillus, Limosilatobacillus, Bifidobacterium, Staphylococcus, Enterococcus, Pseudomonas, Escherichia, Proteus and Salmonella. The degree of stimulation or growth inhibition of tested bacteria depended on the type and dose of the mannoprotein and the bacterial strain. The addition of the tested preparations in the entire range of applied concentrations had a positive effect especially on the growth of Lactobacillus arabinosus ATCC 8014 and Bifidobacterium animalis subsp. lactis B12. Mannoproteins isolated from S. cerevisiae limited the growth of the Escherichia coli (E. coli) ATCC 25922, Pseudomonas aureoginosa (P. aureoginosa) ATCC 27853, Proteus mirabilis ATCC 35659 and Salmonella Enteritidis ATCC 13076 to the greatest extent, while preparations of M. reukaufii and W. anomalus origin most effectively limited the growth of Staphylococcus aureus strains, E. coli and P. aureoginosa. The growth of Enterococcus faecalis was stimulated by the presence of all studied preparations in most of the concentrations used. Further research will determine how the purification process of studied mannoproteins or oligosaccharide fractions, its structure and composition influence on the growth of selected bacteria and what is the mechanism of its activity.

Production of 3-hydroxypropionic acid (3-HP) in Pichia pastoris (syn. Komagataella phaffii) via the malonyl-CoA pathway has been recently demonstrated using glycerol as a carbon source, but the reported metrics were not commercially relevant. The flux through the heterologous pathway from malonyl-CoA to 3-HP was hypothesized as the main bottleneck. In the present study, different metabolic engineering approaches have been combined to improve the productivity of the original 3-HP producing strains. To do so, an additional copy of the gene encoding for the potential rate-limiting step of the pathway, i.e., the C-terminal domain of the malonyl-CoA reductase, was introduced. In addition, a variant of the endogenous acetyl-CoA carboxylase (ACC1S1132A) was overexpressed with the aim to increase the delivery of malonyl-CoA. Furthermore, the genes encoding for the pyruvate decarboxylase, aldehyde dehydrogenase and acetyl-CoA synthase, respectively, were overexpressed to enhance conversion of pyruvate into cytosolic acetyl-CoA, and the main gene responsible for the production of the by-product D-arabitol was deleted. Three different screening conditions were used to classify the performance of the different strains: 24-deep-well plates batch cultures, small-scale cultures in falcon tubes using FeedBeads® (i.e., slow release of glycerol over time), and mini bioreactor batch cultures. The best two strains from the FeedBeads® screening, PpHP8 and PpHP18, were tested in bioreactor fed-batch cultures using a pre-fixed exponentially increasing feeding rate. The strain PpHP18 produced up to 37.05 g L−1 of 3-HP at 0.712 g L−1 h−1 with a final product yield on glycerol of 0.194 Cmol−1 in fed-batch cultures. Remarkably, PpHP18 did not rank among the 2-top producer strains in small scale batch cultivations in deep-well plates and mini bioreactors, highlighting the importance of multiplexed screening conditions for adequate assessment of metabolic engineering strategies. These results represent a 50% increase in the product yield and final concentration, as well as over 30% increase in volumetric productivity compared to the previously obtained metrics for P. pastoris. Overall, the combination of glycerol as carbon source and a metabolically engineered P. pastoris strain resulted in the highest 3-HP concentration and productivity reported so far in yeast.

In industrial settings and processes, yeasts may face multiple adverse environmental conditions. These include exposure to non-optimal temperatures or pH, osmotic stress, and deleterious concentrations of diverse inhibitory compounds. These toxic chemicals may result from the desired accumulation of added-value bio-products, yeast metabolism, or be present or derive from the pre-treatment of feedstocks, as in lignocellulosic biomass hydrolysates. Adaptation and tolerance to industrially relevant stress factors involve highly complex and coordinated molecular mechanisms occurring in the yeast cell with repercussions on the performance and economy of bioprocesses, or on the microbiological stability and conservation of foods, beverages, and other goods. To sense, survive, and adapt to different stresses, yeasts rely on a network of signaling pathways to modulate the global transcriptional response and elicit coordinated changes in the cell. These pathways cooperate and tightly regulate the composition, organization and biophysical properties of the cell wall. The intricacy of the underlying regulatory networks reflects the major role of the cell wall as the first line of defense against a wide range of environmental stresses. However, the involvement of cell wall in the adaptation and tolerance of yeasts to multiple stresses of biotechnological relevance has not received the deserved attention. This article provides an overview of the molecular mechanisms involved in fine-tuning cell wall physicochemical properties during the stress response of Saccharomyces cerevisiae and their implication in stress tolerance. The available information for non-conventional yeast species is also included. These non-Saccharomyces species have recently been on the focus of very active research to better explore or control their biotechnological potential envisaging the transition to a sustainable circular bioeconomy.

Pectin-rich plant biomass residues represent underutilized feedstocks for industrial biotechnology. The conversion of the oxidized monomer D-galacturonic acid (D-GalUA) to highly reduced fermentation products such as alcohols is impossible due to the lack of electrons. The reduced compound glycerol has therefore been considered an optimal co-substrate, and a cell factory able to efficiently co-ferment these two carbon sources is in demand. Here, we inserted the fungal D-GalUA pathway in a strain of the yeast S. cerevisiae previously equipped with an NAD-dependent glycerol catabolic pathway. The constructed strain was able to consume D-GalUA with the highest reported maximum specific rate of 0.23 g gCDW−1 h−1 in synthetic minimal medium when glycerol was added. By means of a 13C isotope-labelling analysis, carbon from both substrates was shown to end up in pyruvate. The study delivers the proof of concept for a co-fermentation of the two ‘respiratory’ carbon sources to ethanol and demonstrates a fast and complete consumption of D-GalUA in crude sugar beet pulp hydrolysate under aerobic conditions. The future challenge will be to achieve co-fermentation under industrial, quasi-anaerobic conditions.

The ascomycetous yeast Candida membranifaciens has been isolated from diverse habitats, including humans, insects, and environmental sources, exhibiting a remarkable ability to use different carbon sources that include pentoses, melibiose, and inulin. In this study, we isolated four C. membranifaciens strains from soil and investigated their potential to overproduce riboflavin. C. membranifaciens IST 626 was found to produce the highest concentrations of riboflavin. The volumetric production of this vitamin was higher when C. membranifaciens IST 626 cells were cultured in a commercial medium without iron and when xylose was the available carbon source compared to the same basal medium with glucose. Supplementation of the growth medium with 2 g/L glycine favored the metabolization of xylose, leading to biomass increase and consequent enhancement of riboflavin volumetric production that reached 120 mg/L after 216 h of cultivation. To gain new insights into the molecular basis of riboflavin production and carbon source utilization in this species, the first annotated genome sequence of C. membranifaciens is reported in this article, as well as the result of a comparative genomic analysis with other relevant yeast species. A total of 5619 genes were predicted to be present in C. membranifaciens IST 626 genome sequence (11.5 Mbp). Among them are genes involved in riboflavin biosynthesis, iron homeostasis, and sugar uptake and metabolism. This work put forward C. membranifaciens IST 626 as a riboflavin overproducer and provides valuable molecular data for future development of superior producing strains capable of using the wide range of carbon sources, which is a characteristic trait of the species.

Among the mechanisms used by yeasts to overcome the deleterious effects of chemical and other environmental stresses is the activity of plasma membrane efflux pumps involved in multidrug resistance (MDR), a role on the focus of intensive research for years in pathogenic yeasts. More recently, these active transporters belonging to the MFS (Drug: H+ antiporters) or the ABC superfamily have been involved in resistance to xenobiotic compounds and in the transport of substrates with a clear physiological role. This review paper focuses on these putative efflux pumps concerning their tolerance phenotypes towards bioprocess-specific multiple stress factors, expression levels, physiological roles, and mechanisms by which they may lead to multistress resistance. Their association with the increased secretion of metabolites and other bioproducts and in the development of more robust superior strains for Yeast Chemical Biotechnology is highlighted.

The species Blastobotrys navarrensis Sesma and Ramirez was delineated based on the description of the single strain CBS 139.77T. Based on its phenotypic similarities to Blastobotrys proliferans, B. navarrensis CBS 139.77T was later considered a synonym of B. proliferans. In the present study, we isolated the yeast strain IST 508 (=PYCC 8784=CBS 16671) from the soil surrounding an olive tree in Ferreira do Alentejo, Portugal. The phylogenetic analysis of D1/D2 domain and ITS sequences from strain IST 508 indicates that is closely related to B. navarrensis and B. proliferans. Although strain IST 508 differs from B. navarrensis CBS 139.77T by 14 substitutions and 20 indels (6.6 % divergence) in the ITS sequence, no divergence was detected at the level of D1/D2 domain, mitochondrial small subunit rDNA, and cytochrome oxidase II sequences. On the other hand, strains IST 508 and CBS 139.77 differ from B. proliferans NRRL Y-17577T by eight substitutions (1.4 % divergence) in the D1/ D2 domain sequence, by 16 substitutions (2.7 % divergence) in the cytochrome oxidase II sequence, and by 16 substitutions (3.7 % divergence) in the mitochondrial small subunit rDNA sequence. Due to the high number of variable phenotypic tests in B. proliferans and B. navarrensis, strains from the two species are difficult to distinguish. Contrasting with what is described for other Blastobotrys species, no differences were detected at the level of micromorphology between the two species. Nevertheless, based on the molecular differences between the two strains, CBS 139.77 and IST 508, and B. proliferans NRRL Y-17577T and their phylogenetic analysis, strains CBS 139.77 and IST 508 are from B. navarrensis and this species should be considered as an independent species and not a later synonym of B. proliferans. We propose an emended description of B. navarrensis.

Exploration of yeast diversity for the sustainable production of biofuels, in particular biodiesel, is gaining momentum in recent years. However, sustainable, and economically viable bioprocesses require yeast strains exhibiting: (i) high tolerance to multiple bioprocess-related stresses, including the various chemical inhibitors present in hydrolysates from lignocellulosic biomass and residues; (ii) the ability to efficiently consume all the major carbon sources present; (iii) the capacity to produce lipids with adequate composition in high yields. More than 160 non-conventional (non-Saccharomyces) yeast species are described as oleaginous, but only a smaller group are relatively well characterised, including Lipomyces starkeyi, Yarrowia lipolytica, Rhodotorula toruloides, Rhodotorula glutinis, Cutaneotrichosporon oleaginosus and Cutaneotrichosporon cutaneum. This article provides an overview of lipid production by oleaginous yeasts focusing on yeast diversity, metabolism, and other microbiological issues related to the toxicity and tolerance to multiple challenging stresses limiting bioprocess performance. This is essential knowledge to better understand and guide the rational improvement of yeast performance either by genetic manipulation or by exploring yeast physiology and optimal process conditions. Examples gathered from the literature showing the potential of different oleaginous yeasts/process conditions to produce oils for biodiesel from agro-forestry and industrial organic residues are provided.

Protein phosphorylation is the most common and versatile post-translational modification occurring in eukaryotes. In yeast, protein phosphorylation is fundamental for maintaining cell growth and adapting to sudden changes in environmental conditions by regulating cellular processes and activating signal transduction pathways. Protein kinases catalyze the reversible addition of phosphate groups to target proteins, thereby regulating their activity. In Saccharomyces cerevisiae, kinases are classified into six major groups based on structural and functional similarities. The NPR/Hal family of kinases comprises nine fungal-specific kinases that, due to lack of similarity with the remaining kinases, were classified to the “Other” group. These kinases are primarily implicated in regulating fundamental cellular processes such as maintaining ion homeostasis and controlling nutrient transporters’ concentration at the plasma membrane. Despite their biological relevance, these kinases remain poorly characterized and explored. This review provides an overview of the information available regarding each of the kinases from the NPR/Hal family, including their known biological functions, mechanisms of regulation, and integration in signaling pathways in S. cerevisiae. Information gathered for non-Saccharomyces species of biotechnological or clinical relevance is also included.

Designing promoter architectures hinges on genomic and functional annotation. Saccharomyces cerevisiae is the first model yeast whose databases host genomic and functional annotation information. To predict transcription factors (TFs) regulating central pathways in yeasts, we first introduce S. cerevisiae cis-acting DNA sequences/sites (cADSs) curation pipeline (Sc-cADSs-CP). The promoters of the genes involved in the central pathways of S. cerevisiae were retrieved from the genome sequences. We processed the binding frequency matrices of TFs with the following two criteria. First, we extracted cADSs based on the TF motifs in the TRANSFAC database; then, if there were more than one frequency matrix for a TF, the longest one with the maximum sensitivity was used. Next, we developed the direct scanning algorithm ScanAlgo (uses the Biopython-library), scanning DNA motifs for pairing cross-species alignments. We used the tools Sc-cADSs-CP and ScanAlgo to predict master TFs for Pichia pastoris, which lacks extensive functional annotation studies. The phylogenetic footprinting results were obtained by aligning the scanned S. cerevisiae promoters against orthologous P. pastoris promoters. The predicted cADSs were summed into position weight matrices unique to P. pastoris. We annotated 116 TFs based on the phylogenetic footprinting predictions of cADSs regulating the central pathways in P. pastoris. The presented methodology with the tools Sc-cADSs-CP and ScanAlgo enables the prediction of master TFs and cADSs in yeasts.

During the conversion of lignocellulose, phenolic wastewaters are generated. Therefore, researchers have investigated wastewater valorization processes in which these pollutants are converted to chemicals, i.e., lipids. However, wastewaters are lean feedstocks, so these valorization processes in research typically require the addition of large quantities of sugars and sterilization, which increase costs. This paper investigates a repeated batch fermentation strategy with Rhodotorula kratochvilovae EXF7516 and Cutaneotrichosporon oleaginosum ATCC 20509, without these requirements. The pollutant removal and its conversion to microbial oil were evaluated. Because of the presence of non-monomeric substrates, the ligninolytic enzyme activity was also investigated. The repeated batch fermentation strategy was successful, as more lipids accumulated every cycle, up to a total of 5.4 g/L (23% cell dry weight). In addition, the yeasts consumed up to 87% of monomeric substrates, i.e., sugars, aromatics, and organics acids, and up to 23% of non-monomeric substrates, i.e., partially degraded xylan, lignin, cellulose. Interestingly, lipid production was only observed during the harvest phase of each cycle, as the cells experienced stress, possibly due to oxygen limitation. This work presents the first results on the feasibility of valorizing non-sterilized lignocellulosic wastewater with R. kratochvilovae and C. oleaginosum using a cost-effective repeated batch strategy.

Odd-chain fatty acids (OCFA) have been studied for their therapeutic and nutritional properties, as well as for their potential use in the chemical industry for the production of biofuel. Genetic modification strategies have demonstrated an improved production of OCFA by oleaginous microorganisms. In this study, the production of OCFA-enriched lipids by fermentation using a genetically engineered Yarrowia lipolytica strain was investigated. The major fatty acid produced by this strain was the cis-9-heptadecenoic acid (C17:1). Its biosynthesis was optimized using a design of experiment strategy involving a central composite design. The optimal responses maximizing the cell density (optical density at 600 nm) and the C17:1 content (%) in lipids were found using 52.4 g/L sucrose, 26.9 g/L glycerol, 10.4 g/L sodium acetate, 5 g/L sodium propionate, and 4 g/L yeast extract. Under these conditions, in a 5 L scale bioreactor, the respective contents of lipids and C17:1 in culture medium were 2.52 ± 0.05 and 0.82 ± 0.01 g/L after 96 h fermentation. The results obtained in this work pave the way toward the process upscale of C17:1 and encourage its industrial production.

Odd-chain fatty acids (OCFAs) have recently gained interest as target compounds in microbial production due to their diverse applications in the medical, pharmaceutical and chemical industries for the production of biofuels. Yarrowia lipolytica is a promising oleaginous yeast that has the ability to accumulate high quantities of fatty acids. However, the use of Y. lipolytica oils is still under research, in order to decrease the production costs related to the fermentation process and improve economic feasibility. In this work, sugar beet molasses (10–50 g/L) and crude glycerol (30 g/L) were used as the main carbon sources to reduce the processing costs of oil production from a genetically engineered Y. lipolytica strain. The effects of medium composition were studied on biomass production, lipid content, and OCFAs profile. Lipid production by yeast growing on molasses (20 g/L sucrose) and crude glycerol reached 4.63 ± 0.95 g/L of culture medium. OCFAs content represented 58% of the total fatty acids in lipids, which corresponds to ≈2.69 ± 0.03 g/L of culture medium. The fermentation was upscaled to 5 L bioreactors and fed-batch co-feeding increased OCFA accumulation in Y. lipolytica by 56% compared to batch cultures. glycerol, 10.4 g/L sodium acetate, 5 g/L sodium propionate, and 4 g/L yeast extract. Under these conditions, in a 5 L scale bioreactor, the respective contents of lipids and C17:1 in culture medium were 2.52 ± 0.05 and 0.82 ± 0.01 g/L after 96 h fermentation. The results obtained in this work pave the way toward the process upscale of C17:1 and encourage its industrial production.

Microbes and their carbohydrate-active enzymes are central for depolymerization of complex lignocellulosic polysaccharides in the global carbon cycle. Their unique abilities to degrade and ferment carbohydrates are also utilized in many industrial processes such as baking, brewing and production of biofuels and drugs. Effective degradation and utilization of cellulose and hemicelluloses is important for the shift towards green bioeconomy, and requires microbes equipped with proper sets of carbohydrate-active enzymes (CAZymes). Knowledge of cellulolytic and xylanolytic CAZymes has mainly been generated from bacteria and filamentous fungi, while yeasts have been largely overlooked and may represent an untapped resource in natural CAZymes with industrial relevance. Cellulose and xylan-degrading yeasts with the ability to ferment saccharides are also promising candidates for consolidated bioprocesses (CBPs), as they can degrade lignocellulose and utilize its constituents to produce desired products at the same time. Cellulolytic yeasts able to utilize insoluble crystalline cellulose are rare while xylanolytic yeasts are rather widespread in nature. The lack of particular enzymes in yeasts can be remediated by introducing the missing enzymes into strains having outstanding product-forming attributes.

In this review, we provide a comprehensive overview of the cellulose- and xylan-degrading ascomycetous and basidiomycetous yeasts known to date. We describe how these yeasts can be identified through bioprospecting and bioinformatic approaches and summarize available growth and enzymatic assays for strain characterization. Known and predicted CAZymes are extensively analyzed, both in individual species and in a phylogenetic perspective. We also describe the strategies used for construction of recombinant cellulolytic and xylanolytic strains as well as current applications for polysaccharide-degrading yeasts. Finally, we discuss the great potential of these yeasts as industrial cell factories, identify open research questions and provide suggestions for future investigations.

Apples are affected by numerous fungi known as storage rots, which cause significant losses before and after harvest. Concerns about increasing antimicrobial resistance, bans on various fungicides, and changing consumer preferences are motivating the search for safer means to prevent fruit rot. The use of antagonistic microbes has been shown to be an efficient and environmentally friendly alternative to conventional phytopharmaceuticals. Here, we investigate the potential of Aureobasidium subglaciale for postharvest rot control. We tested the antagonistic activity of 9 strains of A. subglaciale and 7 closely related strains against relevant phytopathogenic fungi under conditions simulating low-temperature storage: Botrytis cinerea, Penicillium expansum, and Colletotrichum acutatum. We also investigated a selection of phenotypic traits of all strains and sequenced their whole genomes. The tested strains significantly reduced postharvest rot of apples at low temperatures caused by B. cinerea, C. acutatum (over 60%), and P. expansum (about 40%). Several phenotypic traits were observed that may contribute to this biocontrol capacity: growth at low temperatures, tolerance to high temperatures and elevated solute concentrations, and strong production of several extracellular enzymes and siderophores. Population genomics revealed that 7 of the 15 strains originally identified as A. subglaciale most likely belong to other, possibly undescribed species of the same genus. In addition, the population structure and linkage disequilibrium of the species suggest that A. subglaciale is strictly clonal and therefore particularly well suited for use in biocontrol. Overall, these data suggest substantial biological control potential for A. subglaciale, which represents another promising biological agent for disease control in fresh fruit.

Background
The great diversity of lifestyles and survival strategies observed in fungi is reflected in the many ways in which they reproduce and recombine. Although a complete absence of recombination is rare, it has been reported for some species, among them 2 extremotolerant black yeasts from Dothideomycetes: Hortaea werneckii and Aureobasidium melanogenum. Therefore, the presence of diploid strains in these species cannot be explained as the product of conventional sexual reproduction.

Results
Genome sequencing revealed that the ratio of diploid to haploid strains in both H. werneckii and A. melanogenum is about 2:1. Linkage disequilibrium between pairs of polymorphic loci and a high degree of concordance between the phylogenies of different genomic regions confirmed that both species are clonal. Heterozygosity of diploid strains is high, with several hybridizing genome pairs reaching the intergenomic distances typically seen between different fungal species. The origin of diploid strains collected worldwide can be traced to a handful of hybridization events that produced diploids, which were stable over long periods of time and distributed over large geographic areas.

Conclusions
Our results, based on the genomes of over 100 strains of 2 black yeasts, show that although they are clonal, they occasionally form stable and highly heterozygous diploid intraspecific hybrids. The mechanism of these apparently rare hybridization events, which are not followed by meiosis or haploidization, remains unknown. Both extremotolerant yeasts, H. werneckii and even more so A. melanogenum, a close relative of the intensely recombining and biotechnologically relevant Aureobasidium pullulans, provide an attractive model for studying the role of clonality and ploidy in extremotolerant fungi.

Organic acids like succinic and citric acids are of great interest as platform organic products that play important roles as precursors for a wide range of bio-based materials. Succinic and citric acids can be successfully produced biotechnologically from renewable resources of both hydrophilic and hydrophobic nature through efficient microbiological conversion. Yarrowia lipolytica represents one of the most versatile microbial factories in terms of organic acids production, as it easily develops and produced metabolites starting from glucidic-based and/or lipid-based substrates. The purpose of this work was to investigate the ability of Y. lipolytica to adapt to hydrophilic and hydrophobic sources and to biosynthesize important platform chemicals like succinic and citric acids. The selected strain was monitored during a batch cultivation for 192 h on 100 g/L carbon source: pure glycerol as a hydrophilic source, sunflower waste cooking oil as a hydrophobic source, and crude glycerol deriving from biodiesel production as a mixture of hydrophilic and hydrophobic sources. Cellular viability, biomass accumulation, and metabolites formation in terms of succinic acid and citric acid was monitored, and the highest results were registered for cultivations performed on waste cooking oil [10.35 ± 0.29 (log10) CFU/mL, 8.15 g/L cell dry weight, 3.50 ± 0.04 g/L citric acid, and 21 ± 0.16 g/L succinic acid]. The results obtained in this work outline the industrial potential of the oleaginous yeast strain of Y. lipolytica to bioconvert the lipidic residual biomass with negative environmental implications into valuable organic compounds with wide-range applicability.

This work describes a coordinate and comprehensive view on the time course of the alterations occurring at the level of the cell wall during adaptation of a yeast cell population to sudden exposure to a sub-lethal stress induced by acetic acid. Acetic acid is a major inhibitory compound in industrial bioprocesses and a widely used preservative in foods and beverages. Results indicate that yeast cell wall resistance to lyticase activity increases during acetic acid-induced growth latency, corresponding to yeast population adaptation to sudden exposure to this stress. This response correlates with: (i) increased cell stiffness, assessed by atomic force microscopy (AFM); (ii) increased content of cell wall β-glucans, assessed by fluorescence microscopy, and (iii) slight increase of the transcription level of the GAS1 gene encoding a β-1,3-glucanosyltransferase that leads to elongation of (1→3)-β-d-glucan chains. Collectively, results reinforce the notion that the adaptive yeast response to acetic acid stress involves a coordinate alteration of the cell wall at the biophysical and molecular levels. These alterations guarantee a robust adaptive response essential to limit the futile cycle associated to the re-entry of the toxic acid form after the active expulsion of acetate from the cell interior

Halophilic and osmotolerant yeast Debaryomyces hansenii has a high potential for cell factory applications due to its resistance to harsh environmental factors and compatibility with a wide substrate range. However, currently available genetic techniques do not allow the full potential of D. hansenii as a cell factory to be harnessed. Moreover, most of the currently available tools rely on the use of auxotrophic markers that are not suitable in wild-type prototrophic strains. In addition, the preferred non-homologous end-joining (NHEJ) DNA damage repair mechanism poses further challenges when precise gene targeting is required. In this study, we present a novel plasmid-based CRISPRCUG/Cas9 method for easy and efficient gene editing of the prototrophic strains of D. hansenii. Our toolset design is based on a dominant marker and facilitates quick assembly of the vectors expressing Cas9 and single or multiple single-guide RNAs (sgRNAs) that provide the possibility for multiplex gene engineering even in prototrophic strains. Moreover, we have constructed NHEJ-deficient D. hansenii that enable our CRISPRCUG/Cas9 tools to support the highly efficient introduction of point mutations and single/double gene deletions. Importantly, we also demonstrate that 90-nt single-stranded DNA oligonucleotides are sufficient for direct repair of DNA breaks induced by sgRNA-Cas9, resulting in precise edits reaching 100% efficiencies. In conclusion, tools developed in this study will greatly advance basic and applied research in D. hansenii. In addition, we envision that our tools can be rapidly adapted for gene editing of other non-conventional yeast species including the ones belonging to the CUG clade.

Debaryomyces hansenii is a non-conventional yeast considered to be a well-suited option for a number of different industrial bioprocesses. It exhibits a set of beneficial traits (halotolerant, oleaginous, xerotolerant, inhibitory compounds resistant) which translates to a number of advantages for industrial fermentation setups when compared to traditional hosts. Although D. hansenii has been highly studied during the last three decades, especially in regards to its salt-tolerant character, the molecular mechanisms underlying this natural tolerance should be further investigated in order to broadly use this yeast in biotechnological processes. In this work, we performed a series of chemostat cultivations in controlled bioreactors where D. hansenii (CBS 767) was grown in the presence of either 1M NaCl or KCl and studied the transcriptomic and (phospho)proteomic profiles. Our results show that sodium and potassium trigger different responses at both expression and regulation of protein activity levels and also complemented previous reports pointing to specific cellular processes as key players in halotolerance, moreover providing novel information about the specific genes involved in each process. The phosphoproteomic analysis, the first of this kind ever reported in D. hansenii, also implicated a novel and yet uncharacterized cation transporter in the response to high sodium concentrations.