Crosstalk between Yeast Cell Plasma Membrane Ergosterol Content and Cell Wall Stiffness under Acetic Acid Stress Involving Pdr18.

Acetic acid is a major inhibitory compound in several industrial bioprocesses, in particular in lignocellulosic yeast biorefineries. Cell envelope remodeling, involving cell wall and plasma membrane composition, structure and function, is among the mechanisms behind yeast adaptation and tolerance to stress. Pdr18 is a plasma membrane ABC transporter of the pleiotropic drug resistance family and a reported determinant of acetic acid tolerance mediating ergosterol transport. This study provides evidence for the impact of Pdr18 expression in yeast cell wall during adaptation to acetic acid stress. The time-course of acetic-acid-induced transcriptional activation of cell wall biosynthetic genes (FKS1, BGL2, CHS3, GAS1) and of increased cell wall stiffness and cell wall polysaccharide content in cells with the PDR18 deleted, compared to parental cells, is reported. Despite the robust and more intense adaptive response of the pdr18Δ population, the stress-induced increase of cell wall resistance to lyticase activity was below parental strain levels, and the duration of the period required for intracellular pH recovery from acidification and growth resumption was higher in the less tolerant pdr18Δ population. The ergosterol content, critical for plasma membrane stabilization, suffered a drastic reduction in the first hour of cultivation under acetic acid stress, especially in pdr18Δ cells. Results revealed a crosstalk between plasma membrane ergosterol content and cell wall biophysical properties, suggesting a coordinated response to counteract the deleterious effects of acetic acid.

Exploring the biological function of efflux pumps for the development of superior industrial yeasts.

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 N.C.Yeastract and CommunityYeastract databases to study gene and genomic transcription regulation in non-conventional yeasts.

Responding to the recent interest of the yeast research community in non-Saccharomyces cerevisiae species of biotechnological relevance, the N.C.Yeastract (http://yeastract-plus.org/ncyeastract/) was associated to YEASTRACT + (http://yeastract-plus.org/). The YEASTRACT + portal is a curated repository of known regulatory associations between transcription factors (TFs) and target genes in yeasts. N.C.Yeastract gathers all published regulatory associations and TF-binding sites for Komagataellaphaffii (formerly Pichia pastoris), the oleaginous yeast Yarrowia lipolytica, the lactose fermenting species Kluyveromyces lactis and Kluyveromyces marxianus, and the remarkably weak acid-tolerant food spoilage yeast Zygosaccharomyces bailii. The objective of this review paper is to advertise the update of the existing information since the release of N.C.Yeastract in 2019, and to raise awareness in the community about its potential to help the day-to-day work on these species, exploring all the information available in the global YEASTRACT + portal. Using simple and widely used examples, a guided exploitation is offered for several tools: (i) inference of orthologous genes; (ii) search for putative TF binding sites and (iii) inter-species comparison of transcription regulatory networks and prediction of TF-regulated networks based on documented regulatory associations available in YEASTRACT + for well-studied species. The usage potentialities of the new CommunityYeastract platform by the yeast community are also discussed.

From a genome assembly to full regulatory network prediction: the case study of Rhodotorula toruloides putative Haa1-regulon.

Numerous genomes are sequenced and made available to the community through the NCBI portal. However, and, unlike what happens for gene function annotation, annotation of promoter sequences and the underlying prediction of regulatory associations is mostly unavailable, severely limiting the ability to interpret genome sequences in a functional genomics perspective. Here we present an approach where one can download a genome of interest from NCBI in the GenBank Flat File (.gbff) format and, with a minimum set of commands, have all the information parsed, organized and made available through the platform web interface. Also, the new genomes are compared with a given genome of reference in search of homologous genes, shared regulatory elements and predicted transcription associations. We present this approach within the context of Community YEASTRACT of the YEASTRACT + portal, thus benefiting from immediate access to all the comparative genomics queries offered in the YEASTRACT + portal. Besides the yeast community, other communities can install the platform independently, without any constraints. In this work, we exemplify the usefulness of the presented tool, within Community YEASTRACT, in constructing a dedicated database and analysing the genome of the highly promising oleaginous red yeast species Rhodotorula toruloides currently poorly studied at the genome and transcriptome levels and with limited genome editing tools. Regulatory prediction is based on the conservation of promoter sequences and available regulatory networks. The case-study examined is focused on the Haa1 transcription factor-a key regulator of yeast resistance to acetic acid, an important inhibitor of industrial bioconversion of lignocellulosic hydrolysates. The new tool described here led to the prediction of a RtHaa1 regulon with expected impact in the optimization of R. toruloides robustness for lignocellulosic and pectin-rich residue biorefinery processes.

Yeast adaptive response to acetic acid stress involves structural alterations and increased stiffness of the cell wall.

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 ß-glucans, assessed by fluorescence microscopy, and (iii) slight increase of the transcription level of the GAS1 gene encoding a ß-1,3-glucanosyltransferase that leads to elongation of (1→3)-ß-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.

The ABC transporter Pdr18 is required for yeast thermotolerance due to its role in ergosterol transport and plasma membrane properties.

Among the mechanisms by which yeast overcomes multiple stresses is the expression of genes encoding ATP-binding cassette (ABC) transporters required for resistance to a wide range of toxic compounds. These substrates may include weak acids, alcohols, agricultural pesticides, polyamines, metal cations, as in the case of Pdr18. This pleotropic drug resistance transporter was previously proposed to transport ergosterol at the plasma membrane (PM) level contributing to the maintenance of PM lipid organization and reduced diffusional permeation induced by lipophilic compounds. The present work reports a novel phenotype associated with the putative drug/xenobiotic-efflux-pump transporter Pdr18: the resistance to heat shock and to long-term growth at supra-optimal temperatures. Cultivation at 40°C was demonstrated to lead to higher PM permeabilization of a pdr18Δ cell population with the PDR18 gene deleted compared with the parental strain population, as indicated by flow cytometry analysis of propidium iodide stained cells. Cells of pdr18Δ grown at 40°C also exhibited increased transcription levels from genes of the ergosterol biosynthetic pathway, compared with parental cells. However, this adaptive response at 40°C was not enough to maintain PM physiological ergosterol levels in the population lacking the Pdr18 transporter and free ergosterol precursors accumulate in the deletion mutant cells.

Adaptation and Survival of Burkholderia cepacia and B. contaminans During Long-Term Incubation in Saline Solutions Containing Benzalkonium Chloride.

The Burkholderia cepacia complex (Bcc) is a group of opportunistic pathogenic bacteria with a remarkable metabolic capacity and broad genotypic/phenotypic plasticity, allowing their adaptation to hostile conditions, including nutrient depleted solutions containing antimicrobial agents. Bcc bacteria are feared contaminants in pharmaceutical industries and cause nosocomial outbreaks, posing health threats to immunocompromised individuals and cystic fibrosis (CF) patients. In this study, the adaptation and survival of B. cepacia and B. contaminans isolates was investigated after long-term incubation in nutrient depleted saline solutions supplemented with increasing concentrations of the biocidal preservative benzalkonium chloride (BZK), recreating the storage conditions of pharmaceutical products. These epidemiologically related isolates were recovered from intrinsically contaminated saline solutions for nasal application and from two CF patients. Long-term incubation in saline solutions containing BZK led to the development of bacterial sub-populations that survived for at least 16 months, despite an initial 2-3 log decrease in viability, displaying a progressive dose-dependent decrease of colony and cell size, including the appearance of small colony variants (SCVs). Bacterial colonies lost pigmentation, changed the morphotype from rough to smooth and produced more spherical cells during extended incubation with BZK. The development of macroscopically visible cellular aggregates, rich in polysaccharide and harboring viable cells in their interior was triggered by BZK. The existence of a metabolic pathway for BZK degradation was confirmed through genome analysis. This study reveals mechanisms underlying the prevalence of Bcc bacteria as contaminants of pharmaceutical products containing BZK, which often lead to false-negative results during quality control and routine testing.

YEASTRACT+: a portal for cross-species comparative genomics of transcription regulation in yeasts.

The YEASTRACT+ information system (http://YEASTRACT-PLUS.org/) is a wide-scope tool for the analysis and prediction of transcription regulatory associations at the gene and genomic levels in yeasts of biotechnological or human health relevance. YEASTRACT+ is a new portal that integrates the previously existing YEASTRACT (http://www.yeastract.com/) and PathoYeastract (http://pathoyeastract.org/) databases and introduces the NCYeastract (Non-Conventional Yeastract) database (http://ncyeastract.org/), focused on the so-called non-conventional yeasts. The information in the YEASTRACT database, focused on Saccharomyces cerevisiae, was updated. PathoYeastract was extended to include two additional pathogenic yeast species: Candida parapsilosis and Candida tropicalis. Furthermore, the NCYeastract database was created, including five biotechnologically relevant yeast species: Zygosaccharomyces baillii, Kluyveromyces lactis, Kluyveromyces marxianus, Yarrowia lipolytica and Komagataella phaffii. The YEASTRACT+ portal gathers 289 706 unique documented regulatory associations between transcription factors (TF) and target genes and 420 DNA binding sites, considering 247 TFs from 10 yeast species. YEASTRACT+ continues to make available tools for the prediction of the TFs involved in the regulation of gene/genomic expression. In this release, these tools were upgraded to enable predictions based on orthologous regulatory associations described for other yeast species, including two new tools for cross-species transcription regulation comparison, based on multi-species promoter and TF regulatory network analyses.

Physiological Genomics of Multistress Resistance in the Yeast Cell Model and Factory: Focus on MDR/MXR Transporters.

The contemporary approach of physiological genomics is vital in providing the indispensable holistic understanding of the complexity of the molecular targets, signalling pathways and molecular mechanisms underlying the responses and tolerance to stress, a topic of paramount importance in biology and biotechnology. This chapter focuses on the toxicity and tolerance to relevant stresses in the cell factory and eukaryotic model yeast Saccharomyces cerevisiae. Emphasis is given to the function and regulation of multidrug/multixenobiotic resistance (MDR/MXR) transporters. Although these transporters have been considered drug/xenobiotic efflux pumps, the exact mechanism of their involvement in multistress resistance is still open to debate, as highlighted in this chapter. Given the conservation of transport mechanisms from S. cerevisiae to less accessible eukaryotes such as plants, this chapter also provides a proof of concept that validates the relevance of the exploitation of the experimental yeast model to uncover the function of novel MDR/MXR transporters in the plant model Arabidopsis thaliana. This knowledge can be explored for guiding the rational design of more robust yeast strains with improved performance for industrial biotechnology, for overcoming and controlling the deleterious activities of spoiling yeasts in the food industry, for developing efficient strategies to improve crop productivity in agricultural biotechnology.

The Paralogous Genes PDR18 and SNQ2, Encoding Multidrug Resistance ABC Transporters, Derive From a Recent Duplication Event, PDR18 Being Specific to the Saccharomyces Genus.

Pleiotropic drug resistance (PDR) family of ATP-binding cassette (ABC) transporters play a key role in the simultaneous acquisition of resistance to a wide range of structurally and functionally unrelated cytotoxic compounds in yeasts. Saccharomyces cerevisiae Pdr18 was proposed to transport ergosterol at the plasma membrane, contributing to the maintenance of adequate ergosterol content and decreased levels of stress-induced membrane disorganization and permeabilization under multistress challenge leading to resistance to ethanol, acetic acid and the herbicide 2,4-D, among other compounds. PDR18 is a paralog of SNQ2, first described as a determinant of resistance to the chemical mutagen 4-NQO. The phylogenetic and neighborhood analysis performed in this work to reconstruct the evolutionary history of ScPDR18 gene in Saccharomycetaceae yeasts was focused on the 214 Pdr18/Snq2 homologs from the genomes of 117 strains belonging to 29 yeast species across that family. Results support the idea that a single duplication event occurring in the common ancestor of the Saccharomyces genus yeasts was at the origin of PDR18 and SNQ2, and that by chromosome translocation PDR18 gained a subtelomeric region location in chromosome XIV. The multidrug/multixenobiotic phenotypic profiles of S. cerevisiae pdr18Δ and snq2Δ deletion mutants were compared, as well as the susceptibility profile for Candida glabrata snq2Δ deletion mutant, given that this yeast species has diverged previously to the duplication event on the origin of PDR18 and SNQ2 genes and encode only one Pdr18/Snq2 homolog. Results show a significant overlap between ScSnq2 and CgSnq2 roles in multidrug/multixenobiotic resistance (MDR/MXR) as well as some overlap in azole resistance between ScPdr18 and CgSnq2. The fact that ScSnq2 and ScPdr18 confer resistance to different sets of chemical compounds with little overlapping is consistent with the subfunctionalization and neofunctionalization of these gene copies. The elucidation of the real biological role of ScSNQ2 will enlighten this issue. Remarkably, PDR18 is only found in Saccharomyces genus genomes and is present in almost all the recently available 1,000 deep coverage genomes of natural S. cerevisiae isolates, consistent with the relevant encoded physiological function.

Pdr18 is involved in yeast response to acetic acid stress counteracting the decrease of plasma membrane ergosterol content and order.

Saccharomyces cerevisiae has the ability to become less sensitive to a broad range of chemically and functionally unrelated cytotoxic compounds. Among multistress resistance mechanisms is the one mediated by plasma membrane efflux pump proteins belonging to the ABC superfamily, questionably proposed to enhance the kinetics of extrusion of all these compounds. This study provides new insights into the biological role and impact in yeast response to acetic acid stress of the multistress resistance determinant Pdr18 proposed to mediate ergosterol incorporation in plasma membrane. The described coordinated activation of the transcription of PDR18 and of several ergosterol biosynthetic genes (ERG2-4, ERG6, ERG24) during the period of adaptation to acetic acid inhibited growth provides further support to the involvement of Pdr18 in yeast response to maintain plasma membrane ergosterol content in stressed cells. Pdr18 role in ergosterol homeostasis helps the cell to counteract acetic acid-induced decrease of plasma membrane lipid order, increase of the non-specific membrane permeability and decrease of transmembrane electrochemical potential. Collectively, our results support the notion that Pdr18-mediated multistress resistance is closely linked to the status of plasma membrane lipid environment related with ergosterol content and the associated plasma membrane properties.

YEASTRACT: an upgraded database for the analysis of transcription regulatory networks in Saccharomyces cerevisiae.

The YEAst Search for Transcriptional Regulators And Consensus Tracking (YEASTRACT-www.yeastract.com) information system has been, for 11 years, a key tool for the analysis and prediction of transcription regulatory associations at the gene and genomic levels in Saccharomyces cerevisiae. Since its last update in June 2017, YEASTRACT includes approximately 163000 regulatory associations between transcription factors (TF) and target genes in S. cerevisiae, based on more than 1600 bibliographic references; it also includes 247 specific DNA binding consensus recognized by 113 TFs. This release of the YEASTRACT database provides new visualization tools to visualize each regulatory network in an interactive fashion, enabling the user to select and observe subsets of the network such as: (i) considering only DNA binding evidence or both DNA binding and expression evidence; (ii) considering only either positive or negative regulatory associations; or (iii) considering only one set of related environmental conditions. A further tool to observe TF regulons is also offered, enabling a clear-cut understanding of the exact meaning of the available data. We believe that with this new version, YEASTRACT will improve its role as an open web resource instrumental for Yeast Biologists and Systems Biology researchers.

Heterologous expression of the yeast Tpo1p or Pdr5p membrane transporters in Arabidopsis confers plant xenobiotic tolerance.

Soil contamination is a major hindrance for plant growth and development. The lack of effective strategies to remove chemicals released into the environment has raised the need to increase plant resilience to soil pollutants. Here, we investigated the ability of two Saccharomyces cerevisiae plasma-membrane transporters, the Major Facilitator Superfamily (MFS) member Tpo1p and the ATP-Binding Cassette (ABC) protein Pdr5p, to confer Multiple Drug Resistance (MDR) in Arabidopsis thaliana. Transgenic plants expressing either of the yeast transporters were undistinguishable from the wild type under control conditions, but displayed tolerance when challenged with the herbicides 2,4-D and barban. Plants expressing ScTPO1 were also more resistant to the herbicides alachlor and metolachlor as well as to the fungicide mancozeb and the Co2+, Cu2+, Ni2+, Al3+ and Cd2+ cations, while ScPDR5-expressing plants exhibited tolerance to cycloheximide. Yeast mutants lacking Tpo1p or Pdr5p showed increased sensitivity to most of the agents tested in plants. Our results demonstrate that the S. cerevisiae Tpo1p and Pdr5p transporters are able to mediate resistance to a broad range of compounds of agricultural interest in yeast as well as in Arabidopsis, underscoring their potential in future biotechnological applications.

Yeast response and tolerance to benzoic acid involves the Gcn4- and Stp1-regulated multidrug/multixenobiotic resistance transporter Tpo1.

The action of benzoic acid in the food and beverage industries is compromised by the ability of spoilage yeasts to cope with this food preservative. Benzoic acid occurs naturally in many plants and is an intermediate compound in the biosynthesis of many secondary metabolites. The understanding of the mechanisms underlying the response and resistance to benzoic acid stress in the eukaryotic model yeast is thus crucial to design more suitable strategies to deal with this toxic lipophilic weak acid. In this study, the Saccharomyces cerevisiae multidrug transporter Tpo1 was demonstrated to confer resistance to benzoic acid. TPO1 transcript levels were shown to be up-regulated in yeast cells suddenly exposed to this stress agent. This up-regulation is under the control of the Gcn4 and Stp1 transcription factors, involved in the response to amino acid availability, but not under the regulation of the multidrug resistance transcription factors Pdr1 and Pdr3 that have binding sites in TPO1 promoter region. Benzoic acid stress was further shown to affect the intracellular pool of amino acids and polyamines. The observed decrease in the concentration of these nitrogenous compounds, registered upon benzoic acid stress exposure, was not found to be dependent on Tpo1, although the limitation of yeast cells on nitrogenous compounds was found to activate Tpo1 expression. Altogether, the results described in this study suggest that Tpo1 is one of the key players standing in the crossroad between benzoic acid stress response and tolerance and the control of the intracellular concentration of nitrogenous compounds. Also, results can be useful to guide the design of more efficient preservation strategies and the biotechnological synthesis of benzoic acid or benzoic acid-derived compounds.

Increased expression of the yeast multidrug resistance ABC transporter Pdr18 leads to increased ethanol tolerance and ethanol production in high gravity alcoholic fermentation.

BACKGROUND: The understanding of the molecular basis of yeast tolerance to ethanol may guide the design of rational strategies to increase process performance in industrial alcoholic fermentations. A set of 21 genes encoding multidrug transporters from the ATP-Binding Cassette (ABC) Superfamily and Major Facilitator Superfamily (MFS) in S. cerevisiae were scrutinized for a role in ethanol stress resistance. RESULTS: A yeast multidrug resistance ABC transporter encoded by the PDR18 gene, proposed to play a role in the incorporation of ergosterol in the yeast plasma membrane, was found to confer resistance to growth inhibitory concentrations of ethanol. PDR18 expression was seen to contribute to decreased ³H-ethanol intracellular concentrations and decreased plasma membrane permeabilization of yeast cells challenged with inhibitory ethanol concentrations. Given the increased tolerance to ethanol of cells expressing PDR18, the final concentration of ethanol produced during high gravity alcoholic fermentation by yeast cells devoid of PDR18 was lower than the final ethanol concentration produced by the corresponding parental strain. Moreover, an engineered yeast strain in which the PDR18 promoter was replaced in the genome by the stronger PDR5 promoter, leading to increased PDR18 mRNA levels during alcoholic fermentation, was able to attain a 6 % higher ethanol concentration and a 17 % higher ethanol production yield than the parental strain. The improved fermentative performance of yeast cells over-expressing PDR18 was found to correlate with their increased ethanol tolerance and ability to restrain plasma membrane permeabilization induced throughout high gravity fermentation. CONCLUSIONS: PDR18 gene over-expression increases yeast ethanol tolerance and fermentation performance leading to the production of highly inhibitory concentrations of ethanol. PDR18 overexpression in industrial yeast strains appears to be a promising approach to improve alcoholic fermentation performance for sustainable bio-ethanol production.