Cell envelope and stress-responsive pathways underlie an evolved oleaginous Rhodotorula toruloides strain multi-stress tolerance.

BACKGROUND: The red oleaginous yeast Rhodotorula toruloides is a promising cell factory to produce microbial oils and carotenoids from lignocellulosic hydrolysates (LCH). A multi-stress tolerant strain towards four major inhibitory compounds present in LCH and methanol, was derived in our laboratory from strain IST536 (PYCC 5615) through adaptive laboratory evolution (ALE) under methanol and high glycerol selective pressure. RESULTS: Comparative genomic analysis suggested the reduction of the original strain ploidy from triploid to diploid, the occurrence of 21,489 mutations, and 242 genes displaying copy number variants in the evolved strain. Transcriptomic analysis identified 634 genes with altered transcript levels (465 up, 178 down) in the multi-stress tolerant strain. Genes associated with cell surface biogenesis, integrity, and remodelling and involved in stress-responsive pathways exhibit the most substantial alterations at the genome and transcriptome levels. Guided by the suggested stress responses, the multi-stress tolerance phenotype was extended to osmotic, salt, ethanol, oxidative, genotoxic, and medium-chain fatty acid-induced stresses. CONCLUSIONS: The comprehensive analysis of this evolved strain provided the opportunity to get mechanistic insights into the acquisition of multi-stress tolerance and a list of promising genes, pathways, and regulatory networks, as targets for synthetic biology approaches applied to promising cell factories, toward more robust and superior industrial strains. This study lays the foundations for understanding the mechanisms underlying tolerance to multiple stresses in R. toruloides, underscoring the potential of ALE for enhancing the robustness of industrial yeast strains.

The role of ion homeostasis in adaptation and tolerance to acetic acid stress in yeasts.

Maintenance of asymmetric ion concentrations across cellular membranes is crucial for proper yeast cellular function. Disruptions of these ionic gradients can significantly impact membrane electrochemical potential and the balance of other ions, particularly under stressful conditions such as exposure to acetic acid. This weak acid, ubiquitous to both yeast metabolism and industrial processes, is a major inhibitor of yeast cell growth in industrial settings and a key determinant of host colonization by pathogenic yeast. Acetic acid toxicity depends on medium composition, especially on the pH (H+ concentration), but also on other ions' concentrations. Regulation of ion fluxes is essential for effective yeast response and adaptation to acetic acid stress. However, the intricate interplay among ion balancing systems and stress response mechanisms still presents significant knowledge gaps. This review offers a comprehensive overview of the mechanisms governing ion homeostasis, including H+, K+, Zn2+, Fe2+/3+, and acetate, in the context of acetic acid toxicity, adaptation, and tolerance. While focus is given on Saccharomyces cerevisiae due to its extensive physiological characterization, insights are also provided for biotechnologically and clinically relevant yeast species whenever available.

Shared and more specific genetic determinants and pathways underlying yeast tolerance to acetic, butyric, and octanoic acids.

BACKGROUND: The improvement of yeast tolerance to acetic, butyric, and octanoic acids is an important step for the implementation of economically and technologically sustainable bioprocesses for the bioconversion of renewable biomass resources and wastes. To guide genome engineering of promising yeast cell factories toward highly robust superior strains, it is instrumental to identify molecular targets and understand the mechanisms underlying tolerance to those monocarboxylic fatty acids. A chemogenomic analysis was performed, complemented with physiological studies, to unveil genetic tolerance determinants in the model yeast and cell factory Saccharomyces cerevisiae exposed to equivalent moderate inhibitory concentrations of acetic, butyric, or octanoic acids. RESULTS: Results indicate the existence of multiple shared genetic determinants and pathways underlying tolerance to these short- and medium-chain fatty acids, such as vacuolar acidification, intracellular trafficking, autophagy, and protein synthesis. The number of tolerance genes identified increased with the linear chain length and the datasets for butyric and octanoic acids include the highest number of genes in common suggesting the existence of more similar toxicity and tolerance mechanisms. Results of this analysis, at the systems level, point to a more marked deleterious effect of an equivalent inhibitory concentration of the more lipophilic octanoic acid, followed by butyric acid, on the cell envelope and on cellular membranes function and lipid remodeling. The importance of mitochondrial genome maintenance and functional mitochondria to obtain ATP for energy-dependent detoxification processes also emerged from this chemogenomic analysis, especially for octanoic acid. CONCLUSIONS: This study provides new biological knowledge of interest to gain further mechanistic insights into toxicity and tolerance to linear-chain monocarboxylic acids of increasing liposolubility and reports the first lists of tolerance genes, at the genome scale, for butyric and octanoic acids. These genes and biological functions are potential targets for synthetic biology approaches applied to promising yeast cell factories, toward more robust superior strains, a highly desirable phenotype to increase the economic viability of bioprocesses based on mixtures of volatiles/medium-chain fatty acids derived from low-cost biodegradable substrates or lignocellulose hydrolysates.

The Hrk1 kinase is a determinant of acetic acid tolerance in yeast by modulating H+ and K+ homeostasis.

Acetic acid-induced stress is a common challenge in natural environments and industrial bioprocesses, significantly affecting the growth and metabolic performance of Saccharomyces cerevisiae. The adaptive response and tolerance to this stress involves the activation of a complex network of molecular pathways. This study aims to delve deeper into these mechanisms in S. cerevisiae, particularly focusing on the role of the Hrk1 kinase. Hrk1 is a key determinant of acetic acid tolerance, belonging to the NPR/Hal family, whose members are implicated in the modulation of the activity of plasma membrane transporters that orchestrate nutrient uptake and ion homeostasis. The influence of Hrk1 on S. cerevisiae adaptation to acetic acid-induced stress was explored by employing a physiological approach based on previous phosphoproteomics analyses. The results from this study reflect the multifunctional roles of Hrk1 in maintaining proton and potassium homeostasis during different phases of acetic acid-stressed cultivation. Hrk1 is shown to play a role in the activation of plasma membrane H+-ATPase, maintaining pH homeostasis, and in the modulation of plasma membrane potential under acetic acid stressed cultivation. Potassium (K+) supplementation of the growth medium, particularly when provided at limiting concentrations, led to a notable improvement in acetic acid stress tolerance of the hrk1Δ strain. Moreover, abrogation of this kinase expression is shown to confer a physiological advantage to growth under K+ limitation also in the absence of acetic acid stress. The involvement of the alkali metal cation/H+ exchanger Nha1, another proposed molecular target of Hrk1, in improving yeast growth under K+ limitation or acetic acid stress, is proposed.

An Evolved Strain of the Oleaginous Yeast Rhodotorula toruloides, Multi-Tolerant to the Major Inhibitors Present in Lignocellulosic Hydrolysates, Exhibits an Altered Cell Envelope.

The presence of toxic compounds in lignocellulosic hydrolysates (LCH) is among the main barriers affecting the efficiency of lignocellulose-based fermentation processes, in particular, to produce biofuels, hindering the production of intracellular lipids by oleaginous yeasts. These microbial oils are promising sustainable alternatives to vegetable oils for biodiesel production. In this study, we explored adaptive laboratory evolution (ALE), under methanol- and high glycerol concentration-induced selective pressures, to improve the robustness of a Rhodotorula toruloides strain, previously selected to produce lipids from sugar beet hydrolysates by completely using the major C (carbon) sources present. An evolved strain, multi-tolerant not only to methanol but to four major inhibitors present in LCH (acetic acid, formic acid, hydroxymethylfurfural, and furfural) was isolated and the mechanisms underlying such multi-tolerance were examined, at the cellular envelope level. Results indicate that the evolved multi-tolerant strain has a cell wall that is less susceptible to zymolyase and a decreased permeability, based on the propidium iodide fluorescent probe, in the absence or presence of those inhibitors. The improved performance of this multi-tolerant strain for lipid production from a synthetic lignocellulosic hydrolysate medium, supplemented with those inhibitors, was confirmed.

YEASTRACT+: a portal for the exploitation of global transcription regulation and metabolic model data in yeast biotechnology and pathogenesis.

YEASTRACT+ (http://yeastract-plus.org/) is a tool for the analysis, prediction and modelling of transcription regulatory data at the gene and genomic levels in yeasts. It incorporates three integrated databases: YEASTRACT (http://yeastract-plus.org/yeastract/), PathoYeastract (http://yeastract-plus.org/pathoyeastract/) and NCYeastract (http://yeastract-plus.org/ncyeastract/), focused on Saccharomyces cerevisiae, pathogenic yeasts of the Candida genus, and non-conventional yeasts of biotechnological relevance. In this release, YEASTRACT+ offers upgraded information on transcription regulation for the ten previously incorporated yeast species, while extending the database to another pathogenic yeast, Candida auris. Since the last release of YEASTRACT+ (January 2020), a fourth database has been integrated. CommunityYeastract (http://yeastract-plus.org/community/) offers a platform for the creation, use, and future update of YEASTRACT-like databases for any yeast of the users' choice. CommunityYeastract currently provides information for two Saccharomyces boulardii strains, Rhodotorula toruloides NP11 oleaginous yeast, and Schizosaccharomyces pombe 972h-. In addition, YEASTRACT+ portal currently gathers 304 547 documented regulatory associations between transcription factors (TF) and target genes and 480 DNA binding sites, considering 2771 TFs from 11 yeast species. A new set of tools, currently implemented for S. cerevisiae and C. albicans, is further offered, combining regulatory information with genome-scale metabolic models to provide predictions on the most promising transcription factors to be exploited in cell factory optimisation or to be used as novel drug targets. The expansion of these new tools to the remaining YEASTRACT+ species is ongoing.

The NPR/Hal family of protein kinases in yeasts: biological role, phylogeny and regulation under environmental challenges.

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.

The cell wall and the response and tolerance to stresses of biotechnological relevance in yeasts.

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.

Exploring Yeast Diversity to Produce Lipid-Based Biofuels from Agro-Forestry and Industrial Organic Residues.

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, Cutaneotrichosporonoleaginosus 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.

Vibriosis Outbreaks in Aquaculture: Addressing Environmental and Public Health Concerns and Preventive Therapies Using Gilthead Seabream Farming as a Model System.

Bacterial and viral diseases in aquaculture result in severe production and economic losses. Among pathogenic bacteria, species belonging to the Vibrio genus are one of the most common and widespread disease-causing agents. Vibrio infections play a leading role in constraining the sustainable growth of the aquaculture sector worldwide and, consequently, are the target of manifold disease prevention strategies. During the early, larval stages of development, Vibrio species are a common cause of high mortality rates in reared fish and shellfish, circumstances under which the host organisms might be highly susceptible to disease preventive or treatment strategies such as vaccines and antibiotics use, respectively. Regardless of host developmental stage, Vibrio infections may occur suddenly and can lead to the loss of the entire population reared in a given aquaculture system. Furthermore, the frequency of Vibrio-associated diseases in humans is increasing globally and has been linked to anthropic activities, in particular human-driven climate change and intensive livestock production. In this context, here we cover the current knowledge of Vibrio infections in fish aquaculture, with a focus on the model species gilthead seabream (Sparus aurata), a highly valuable reared fish in the Mediterranean climatic zone. Molecular methods currently used for fast detection and identification of Vibrio pathogens and their antibiotic resistance profiles are addressed. Targeted therapeutic approaches are critically examined. They include vaccination, phage therapy and probiotics supplementation, which bear promise in supressing vibriosis in land-based fish rearing and in mitigating possible threats to human health and the environment. This literature review suggests that antibiotic resistance is increasing among Vibrio species, with the use of probiotics constituting a promising, sustainable approach to prevent Vibrio infections in aquaculture.

Prediction of Gene and Genomic Regulation in Candida Species, Using the PathoYeastract Database: A Comparative Genomics Approach.

The ability of living organisms to survive changing environmental conditions is dependent on the implementation of gene expression programs underlying adaptation and fitness. Transcriptional networks can be exceptionally complex: a single transcription factor (TF) may regulate hundreds of genes, and multiple TFs may regulate a single gene-depending on the environmental conditions. Moreover, the same TF may act as an activator or repressor in distinct conditions. In turn, the activity of regulators themselves may be dependent on other TFs, as well as posttranscriptional and posttranslational regulation. These traits greatly contribute to the intricate networks governing gene expression programs.In this chapter, a step-by-step guide of how to use PathoYeastract, one of several interconnecting databases within the YEASTRACT+ portal, to predict gene and genomic regulation in Candida spp. is provided. PathoYeastract contains a set of analysis tools to study regulatory associations in human pathogenic yeasts, enabling: (1) the prediction and ranking of TFs that contribute to the regulation of individual genes; (2) the prediction of the genes regulated by a given TF; and (3) the prediction and ranking of TFs that regulate a genome-wide transcriptional response. These capabilities are illustrated, respectively, with the analysis of: (1) the TF network controlling the C. glabrata QDR2 gene; (2) the regulon controlled by the C. glabrata TF Rpn4; and (3) the regulatory network controlling the C. glabrata transcriptome-wide changes induced upon exposure to the antifungal drug fluconazole. The newest potentialities of this information system are explored, including cross-species network comparison. The results are discussed considering the performed queries and integrated with the current knowledge on the biological data for each case-study.

Characterization of a new Blastobotrys navarrensis strain indicates that it is not a later synonym of Blastobotrys proliferans.

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.

Genome Sequence and Analysis of the Flavinogenic Yeast Candida membranifaciens IST 626.

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.

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.

Towards valorization of pectin-rich agro-industrial residues: Engineering of Saccharomyces cerevisiae for co-fermentation of d-galacturonic acid and glycerol.

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.

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.