Potential of natamycin to control growth of Zygosaccharomyces spp. in apple juice during storage.

In this research, anti-yeast activity of natamycin in apple juice inoculated with both Zygosaccharomyces rouxii and Z. bailii during the storage at different temperatures was investigated. For this purpose, a response surface methodology approach was used to test and optimize effects of some processing variables; storage time (1, 21 and 41 days), storage temperature (4, 12 and 20 °C), sodium benzoate as a positive control (0, 0.05 and 0.1%) and natamycin concentration (0, 30 and 60 mg/L) on several physicochemical and bioactive properties of the apple juice samples. The results showed that the natamycin performed a remarkable anti-yeast effect on Z. bailii rather than on Z. rouxii. The brix levels of the samples decreased and so the turbidity values increased significantly due to the yeast activity during the storage. Bioactive properties were also significantly affected by the natamycin which was also revealed to increase the antioxidant capacity of apple juice during storage. Using multiple response optimization technique, it was calculated that minimum yeast count (YC) values would occur at storage time = 38.64 and 40.9 days, storage temperature = 19.81 and 14.4 °C, sodium benzoate level (fixed to 0%) and natamycin concentration = 40 and 51.9 mg/L for the samples inoculated with Z. bailii and Z. rouxii, respectively. It was concluded that the bioactive properties of apple juice could be preserved by addition of natamycin which is suggested to be a natural inhibitor during the storage.

Zygosaccharomyces rouxii Combats Salt Stress by Maintaining Cell Membrane Structure and Functionality.

Zygosaccharomyces rouxii is an important yeast that is required in the food fermentation process due to its high salt tolerance. In this study, the responses and resistance strategies of Z. rouxii against salt stress were investigated by performing physiological analysis at membrane level. The results showed that under salt stress, cell integrity was destroyed, and the cell wall was ruptured, which was accompanied by intracellular substance spillover. With an increase of salt concentrations, intracellular Na+ content increased slightly, whereas intracellular K+ content decreased significantly, which caused the increase of the intracellular Na+/K+ ratio. In addition, in response to salt stress, the activity of Na+/K+-ATPase increased from 0.54 to 2.14 µmol/mg protein, and the ergosterol content increased to 2.42-fold to maintain membrane stability. Analysis of cell membrane fluidity and fatty acid composition showed that cell membrane fluidity decreased and unsaturated fatty acid proportions increased, leading to a 101.21% rise in the unsaturated/saturated fatty acid ratio. The results presented in this study offer guidance in understanding the salt tolerance mechanism of Z. rouxii, and in developing new strategies to increase the industrial utilization of this species under salt stress.

A set of plasmids carrying antibiotic resistance markers and Cre recombinase for genetic engineering of nonconventional yeast Zygosaccharomyces rouxii.

The so-called nonconventional yeasts are becoming increasingly attractive in food and industrial biotechnology. Among them, Zygosaccharomyces rouxii is known to be halotolerant, osmotolerant, petite negative, and poorly Crabtree positive. These traits and the high fermentative vigour make this species very appealing for industrial and food applications. Nevertheless, the biotechnological exploitation of Z. rouxii has been biased by the low availability of genetic engineering tools and the recalcitrance of this yeast towards the most conventional transformation procedures. Centromeric and episomal Z. rouxii plasmids have been successfully constructed with prototrophic markers, which limited their usage to auxotrophic strains, mainly derived from the Z. rouxii haploid type strain Centraalbureau voor Schimmelcultures (CBS) 732T . By contrast, the majority of industrially promising Z. rouxii yeasts are prototrophic and allodiploid/aneuploid strains. In order to expand the genetic tools for manipulating these strains, we developed two centromeric and two episomal vectors harbouring KanMXR and ClonNATR as dominant drug resistance markers, respectively. We also constructed the plasmid pGRCRE that allows the Cre recombinase-mediated marker recycling during multiple gene deletions. As proof of concept, pGRCRE was successfully used to rescue the kanMX-loxP module in Z. rouxii ATCC 42981 G418-resistant mutants previously constructed by replacing the MATαP expression locus with the loxP-kanMX-loxP cassette.

Zygosaccharomyces bailii and Z. rouxii induced ethanol formation in apple juice supplemented with different natural preservatives: A response surface methodology approach.

In this study, ethanol produced by osmophilic yeasts, Zygosaccharomyces bailii and Z. rouxii, in apple juice preserved with mint essential oil (MEO), carvacrol and natamycin instead of synthetic preservatives was modeled. Some processing parameters such as sodium benzoate (SB, 0-0.1%) used as a positive control, storage temperature (4-20 °C) and storage time (1-41 days) were selected in the study. Box-Behnken design in response surface methodology was used to evaluate the effects of processing parameters on ethanol levels of apple juice and three models were created for three preservatives for each yeast. Preservative type affected the ethanol formation in apple juice for both yeasts studied. Increase of preservative concentration decreased the ethanol formation during the storage period. The best effective preservative was determined as MEO and Z. bailii was found to be quite resistant yeast against to the preserving agents for three models as compared to Z. rouxii. Ethanol level increased with the increase of both storage temperature and time for both yeasts. The results showed that apple juice could be preserved by these three preservatives, but the MEO was the most effective agent for apple juice during the storage.

Effect of oregano (Origanum vulgare L. ssp. hirtum) and clove (Eugenia spp.) nanoemulsions on Zygosaccharomyces bailii survival in salad dressings.

This work aimed to evaluate the in vitro effect of encapsulated oregano and clove essential oils on oil-in-water nanoemulsions against Zygosaccharomyces bailii. The antifungal efficacy of these nanoemulsions and their sensory acceptance were tested in salad dressings. Both essential oils were effective inhibitors against the target yeast, with minimal inhibitory and fungicidal concentrations of 1.75 mg/mL. In the in vitro assay done with the nanoemulsions, no yeast growth was observed for any tested essential oil concentration. In the salad dressings, all the formulations were able to reduce Z. bailii growth compared to the control, and only those samples with 1.95 mg/g of essential oil were capable of inhibiting yeast development after 4 inoculation days. The sensory acceptance of the dressing containing the nanoemulsions was similar to the control dressing in appearance, consistency and colour terms. These results evidence the antifungal activity of oregano and clove nanoemulsions against Z. bailii.

Physiological Genomics of the Highly Weak-Acid-Tolerant Food Spoilage Yeasts of Zygosaccharomyces bailii sensu lato.

Zygosaccharomyces bailii and two closely related species, Z. parabailii and Z. pseudobailii ("Z. bailii species complex", "Z. bailii sensu lato" or simply "Z. bailii (s.l.)"), are frequently implicated in the spoilage of acidified preserved foods and beverages due to their tolerance to very high concentrations of weak acids used as food preservatives. The recent sequencing and annotation of these species' genomes have clarified their genomic organization and phylogenetic relationship, which includes events of interspecies hybridization. Mechanistic insights into their adaptation and tolerance to weak acids (e.g., acetic and lactic acids) are also being revealed. Moreover, the potential of Z. bailii (s.l.) to be used in industrial biotechnological processes as interesting cell factories for the production of organic acids, reduction of the ethanol content, increase of alcoholic beverages aroma complexity, as well as of genetic source for increasing weak acid resistance in yeast, is currently being considered. This chapter includes taxonomical, ecological, physiological, and biochemical aspects of Z. bailii (s.l.). The focus is on the exploitation of physiological genomics approaches that are providing the indispensable holistic knowledge to guide the effective design of strategies to overcome food spoilage or the rational exploitation of these yeasts as promising cell factories.

Modelling growth/no growth interface of Zygosaccharomyces bailii in simulated acid sauces as a function of natamycin, xanthan gum and sodium chloride concentrations.

Probabilistic microbial modelling using logistic regression was used to predict the growth/no growth (G/NG) interfaces of Zygosaccharomyces bailii in simulated acid sauces as a function of natamycin, xanthan gum (XG) and sodium chloride concentrations. The growth was assessed colorimetrically by using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride and 2-methoxy-1,4-naphthoquinone as detection reagents. The logistic regression model successfully predicted G/NG probability. The detection reagents used allowed the evaluation of G/NG interfaces in opaque systems with an excellent agreement with the plate count method. Natamycin concentration of 12 mg/L was needed to inhibit Z. bailii growth independently of the presence of XG and/or NaCl. Addition of 3.00 and 6.00% of NaCl exerted an antagonistic effect on natamycin action. Furthermore, addition of 0.25 and 0.50% XG decreased natamycin and/or NaCl action. However, an increased in XG concentration to 1.00% decreased yeast growth. Mentioned results highlighted the importance of the correct selection of stress factors applied to inhibit Z. bailii growth.

Effect of anthocyanins on lipid oxidation and microbial spoilage in value-added emulsions with bilberry seed oil, anthocyanins and cold set whey protein hydrogels.

The objective of this work was to explore the storage properties of a structured oil-in-water emulsion containing both water- and fat-soluble bioactive compounds from bilberries (Vaccinium myrtillus L.). Bilberry seed oil (BSO) was dispersed in a continuous aqueous phase of anthocyanins (AC) and whey protein isolate. The microstructure was evaluated using light microscopy and the effect of anthocyanins on lipid oxidation and microbial growth was investigated. The results showed that it was possible to generate a stable emulsion structure that resisted phase separation during 25 weeks of storage. Gas chromatography-mass spectrometry measurements of the fatty acids in the BSO during storage showed that AC had a protective effect against lipid oxidation. The AC did not have an antimicrobial effect against the investigated strains Zygosaccharomyces bailii (ATCC 42476) and Aspergillus niger (ATCC 6275 (M68)).

Transcriptional profiling of Zygosaccharomyces bailii early response to acetic acid or copper stress mediated by ZbHaa1.

The non-conventional yeast species Zygosaccharomyces bailii is remarkably tolerant to acetic acid, a highly important microbial inhibitory compound in Food Industry and Biotechnology. ZbHaa1 is the functional homologue of S. cerevisiae Haa1 and a bifunctional transcription factor able to modulate Z. bailii adaptive response to acetic acid and copper stress. In this study, RNA-Seq was used to investigate genomic transcription changes in Z. bailii during early response to sublethal concentrations of acetic acid (140 mM, pH 4.0) or copper (0.08 mM) and uncover the regulatory network activated by these stresses under ZbHaa1 control. Differentially expressed genes in response to acetic acid exposure (297) are mainly related with the tricarboxylic acid cycle, protein folding and stabilization and modulation of plasma membrane composition and cell wall architecture, 17 of which, directly or indirectly, ZbHaa1-dependent. Copper stress induced the differential expression of 190 genes mainly involved in the response to oxidative stress, 15 ZbHaa1-dependent. This study provides valuable mechanistic insights regarding Z. bailii adaptation to acetic acid or copper stress, as well as useful information on transcription regulatory networks in pre-whole genome duplication (WGD) (Z. bailii) and post-WGD (S. cerevisiae) yeast species, contributing to the understanding of transcriptional networks' evolution in yeasts.

Effects of dielectric barrier discharge plasma on the inactivation of Zygosaccharomyces rouxii and quality of apple juice.

This work aimed to evaluate the effects of dielectric barrier discharge (DBD) plasma on inactivation of spoilage yeast Zygosaccharomyces rouxii (Z. rouxii), in apple juice. Results showed that DBD plasma treatment at 90 W for 140 s resulted in about 5-log reduction of Z. rouxii in apple juice. The levels of extracellular nucleic acids and proteins as well as contents of H2O2 and NO2- in yeast extract-peptone-dextrose (YPD) medium increased significantly after DBD plasma treatment at 90 W for 40-200 s. The increases in membrane permeability and generation of reactive species would likely contribute to DBD plasma-mediated inactivation of Z. rouxii. DBD plasma caused significant changes in pH, titratable acidity, and certain color parameters of apple juice, but had no effect on the contents of total soluble solids, reducing sugar, and total phenolics. This study provides key implications for the application of DBD plasma in fruit juice processing.

Preservative effect of Camellia sinensis (L.) Kuntze seed extract in soy sauce and its mutagenicity.

The purpose of this study was to investigate the applicability of green tea seed (GTS) extract as a natural preservative in food. Food preservative ability and mutagenicity studies of GTS extract and identification of antimicrobial compounds from GTS extract were carried out. The GTS extract showed only anti-yeast activity against Candida albicans with MIC value of 938µg/mL and Zygosaccharomyces rouxii with a MIC of 469µg/mL. The active compounds were identified as theasaponin E1 (1), assamsaponin A (2), and assamsaponin B (3). And GTS extracts didn't show mutagenicity because there were no dose-dependent changes in colonies of Salmonella typhimurium TA98, TA100, TA1535, TA1537, and Escherichia coli WP2uvrA regardless of the metabolic activation system. And GTS extract also showed a potent food preservation affect which eliminated all yeast below the MIC value in application test at soy sauce. Overall, these results indicate that GTS extract could be a safe and effective food preservative with anti-yeast activity.

Selection of Zygosaccharomyces rouxii strains resistant to cadmium with improved removal abilities through ultraviolet-diethyl sulfate cooperative mutagenesis.

Cd2+ resistance and bioaccumulation capacity were selected from parental Zygosaccharomyces rouxii (CRZ-0) while maintaining NaCl tolerance using protoplast mutagenesis technology. Ultraviolet-diethyl sulfate (UV-DES) cooperative mutagenesis, followed by preliminary screening and rescreening, was used to select the mutant strain CRZ-9. CRZ-9 grew better than CRZ-0 in YPD medium with 20 or 50 mg L-1 of Cd2+. Scanning electron microscopy observations and flow cytometry tests indicated that CRZ-9 was more effective at eliminating reactive oxygen species (ROS) generated by Cd2+, which led to less cellular structural damage and lower lethality. Furthermore, compared with CRZ-0, CRZ-9 exhibited increased potential for application with higher Cd2+ removal ratio, wider working pH range, and lower biomass dosage in Cd2+ bioaccumulation. The mutant strain CRZ-9 possessed improved Cd2+ resistance and bioaccumulation capacity and therefore is a promising strain to remove Cd2+ from wastewater.

The spoilage yeast Zygosaccharomyces bailii: Foe or friend?

Zygosaccharomyces bailii is a non-Saccharomyces budding yeast known as one of the most aggressive food spoilage microorganisms, often isolated as a contaminant during wine fermentation, as well as from many acidic, high-sugar and canned foods. The spoilage ability relies on the yeast's unique feature of tolerating the most common preservatives such as sulphite, dimethyl dicarbonate, acetic acid and sorbic acid. Therefore, many studies have focused on the description of this peculiar tolerance with the aim of developing preventative measures against Z. bailii food spoilage. These studies demonstrated the involvement of diverse molecular and physiological mechanisms in the yeast resistance, comprising detoxification of preservatives, adaptation of the cytoplasmic pH and modulation of the cell wall/membrane composition. At the same time, the described traits unveiled Z. bailii as a novel potential workhorse for industrial bioprocesses. Here we present the yeast Z. bailii starting from important aspects of its robustness and concluding with the exploitation of its potential in biotechnology. Overall, the article describes Z. bailii from different perspectives, converging in presenting it as one of the most interesting species of the Saccharomycotina subphylum. Copyright © 2017 John Wiley & Sons, Ltd.

Biotechnological exploitation of Tetrapisispora phaffii killer toxin: heterologous production in Komagataella phaffii (Pichia pastoris).

The use of natural antimicrobials from plants, animals and microorganisms to inhibit the growth of pathogenic and spoilage microorganisms is becoming more frequent. This parallels the increased consumer interest towards consumption of minimally processed food and 'greener' food and beverage additives. Among the natural antimicrobials of microbial origin, the killer toxin produced by the yeast Tetrapisispora phaffii, known as Kpkt, appears to be a promising natural antimicrobial agent. Kpkt is a glycoprotein with ß-1,3-glucanase and killer activity, which induces ultrastructural modifications to the cell wall of yeast of the genera Kloeckera/Hanseniaspora and Zygosaccharomyces. Moreover, Kpkt maintains its killer activity in grape must for at least 14 days under winemaking conditions, thus suggesting its use against spoilage yeast in wine making and the sweet beverage industry. Here, the aim was to explore the possibility of high production of Kpkt for biotechnological exploitation. Molecular tools for heterologous production of Kpkt in Komagataella phaffii GS115 were developed, and two recombinant clones that produce up to 23 mg/L recombinant Kpkt (rKpkt) were obtained. Similar to native Kpkt, rKpkt has ß-glucanase and killer activities. Moreover, it shows a wider spectrum of action with respect to native Kpkt. This includes effects on Dekkera bruxellensis, a spoilage yeast of interest not only in wine making, but also for the biofuel industry, thus widening the potential applications of this rKpkt.

Culture medium optimization for osmotolerant yeasts by use of a parallel fermenter system and rapid microbiological testing.

In the present study, a culture medium for qualitative detection of osmotolerant yeasts, named OM, was developed. For the development, culture media with different concentrations of glucose, fructose, potassium chloride and glycerin were analyzed in a Biolumix™ test incubator. Selectivity for osmotolerant yeasts was guaranteed by a water activity (aw)-value of 0.91. The best results regarding fast growth of Zygosaccharomyces rouxii (WH 1002) were achieved in a culture medium consisting of 45% glucose, 5% fructose and 0.5% yeast extract and in a medium with 30% glucose, 10% glycerin, 5% potassium chloride and 0.5% yeast extract. Substances to stimulate yeast fermentation rates were analyzed in a RAMOS® parallel fermenter system, enabling online measurement of the carbon dioxide transfer rate (CTR) in shaking flasks. Significant increases of the CTR was achieved by adding especially 0.1-0.2% ammonium salts ((NH4)2HPO4, (NH4)2SO4 or NH4NO3), 0.5% meat peptone and 1% malt extract. Detection times and the CTR of 23 food-borne yeast strains of the genera Zygosaccharomyces, Torulaspora, Schizosaccharomyces, Candida and Wickerhamomyces were analyzed in OM bouillon in comparison to the selective culture media YEG50, MYG50 and DG18 in the parallel fermenter system. The OM culture medium enabled the detection of 102CFU/g within a time period of 2-3days, depending on the analyzed yeast species. Compared with YEG50 and MYG50 the detection times could be reduced. As an example, W. anomalus (WH 1021) was detected after 124h in YEG50, 95.5h in MYG50 and 55h in OM bouillon. Compared to YEG50 the maximum CO2 transfer rates for Z. rouxii (WH 1001), T. delbrueckii (DSM 70526), S. pombe (DSM 70576) and W. anomalus (WH 1016) increased by a factor ≥2.6. Furthermore, enrichment cultures of inoculated high-sugar products in OM culture medium were analyzed in the Biolumix™ system. The results proved that detection times of 3days for Z. rouxii and T. delbrueckii can be realized by using OM in combination with the automated test system even if low initial counts (101CFU/g) are present in the products. In conclusion, the presented data suggest that the OM culture medium is appropriate for the enrichment of osmotolerant yeasts from high-sugar food products.

Differential hypersaline stress response in Zygosaccharomyces rouxii complex yeasts: a physiological and transcriptional study.

The Zygosaccharomyces rouxii complex comprises three distinct lineages of halotolerant yeasts relevant in food processing and spoilage, such as Z. sapae, Z. rouxii and a mosaic group of allodiploid strains. They manifest plastic genome architecture (variation in karyotype, ploidy level and Na(+)/H(+) antiporter-encoding gene copy number), and exhibit diverse tolerances to salt concentrations. Here, we investigated accumulation of compatible osmolytes and transcriptional regulation of Na(+)/H(+) antiporter-encoding ZrSOD genes during salt exposure in strains representative for the lineages, namely Z. sapae ABT301(T) (low salt tolerant), Z. rouxii CBS 732(T) (middle salt tolerant) and allodiploid strain ATCC 42981 (high salt tolerant). Growth curve modelling in 2 M NaCl-containing media supplemented with or without yeast extract as nitrogen source indicates that moderate salt tolerance of CBS 732(T) mainly depends on nitrogen availability rather than intrinsic inhibitory effects of salt. All the strains produce glycerol and not mannitol under salt stress and use two different glycerol balance strategies. ATCC 42981 produces comparatively more glycerol than Z. sapae and Z. rouxii under standard growth conditions and better retains it intracellularly under salt injuries. Conversely, Z. sapae and Z. rouxii enhance glycerol production under salt stress and intracellularly retain glycerol less efficiently than ATCC 42981. Expression analysis shows that, in diploid Z. sapae and allodiploid ATCC 42981, transcription of gene variants ZrSOD2-22/ZrSOD2 and ZrSOD22 is constitutive and salt unresponsive.

Assessing physio-macromolecular effects of lactic acid on Zygosaccharomyces bailii cells during microaerobic fermentation.

The ability of Zygosaccharomyces bailii to grow at low pH and in the presence of considerable amounts of weak organic acids, at lethal condition for Saccharomyces cerevisiae, increased the interest in the biotechnological potential of the yeast. To understand the mechanism of tolerance and growth effect of weak acids on Z. bailii, we evaluated the physiological and macromolecular changes of the yeast exposed to sub lethal concentrations of lactic acid. Lactic acid represents one of the important commodity chemical which can be produced by microbial fermentation. We assessed physiological effect of lactic acid by bioreactor fermentation using synthetic media at low pH in the presence of lactic acid. Samples collected from bioreactors were stained with propidium iodide (PI) which revealed that, despite lactic acid negatively influence the growth rate, the number of PI positive cells is similar to that of the control. Moreover, we have performed Fourier Transform Infra-Red (FTIR) microspectroscopy analysis on intact cells of the same samples. This technique has been never applied before to study Z. bailii under this condition. The analyses revealed lactic acid induced macromolecular changes in the overall cellular protein secondary structures, and alterations of cell wall and membrane physico-chemical properties.

Sphingolipids contribute to acetic acid resistance in Zygosaccharomyces bailii.

Lignocellulosic raw material plays a crucial role in the development of sustainable processes for the production of fuels and chemicals. Weak acids such as acetic acid and formic acid are troublesome inhibitors restricting efficient microbial conversion of the biomass to desired products. To improve our understanding of weak acid inhibition and to identify engineering strategies to reduce acetic acid toxicity, the highly acetic-acid-tolerant yeast Zygosaccharomyces bailii was studied. The impact of acetic acid membrane permeability on acetic acid tolerance in Z. bailii was investigated with particular focus on how the previously demonstrated high sphingolipid content in the plasma membrane influences acetic acid tolerance and membrane permeability. Through molecular dynamics simulations, we concluded that membranes with a high content of sphingolipids are thicker and more dense, increasing the free energy barrier for the permeation of acetic acid through the membrane. Z. bailii cultured with the drug myriocin, known to decrease cellular sphingo-lipid levels, exhibited significant growth inhibition in the presence of acetic acid, while growth in medium without acetic acid was unaffected by the myriocin addition. Furthermore, following an acetic acid pulse, the intracellular pH decreased more in myriocin-treated cells than in control cells. This indicates a higher inflow rate of acetic acid and confirms that the reduction in growth of cells cultured with myriocin in the medium with acetic acid was due to an increase in membrane permeability, thereby demonstrating the importance of a high fraction of sphingolipids in the membrane of Z. bailii to facilitate acetic acid resistance; a property potentially transferable to desired production organisms suffering from weak acid stress.

Search for genes responsible for the remarkably high acetic acid tolerance of a Zygosaccharomyces bailii-derived interspecies hybrid strain.

BACKGROUND: Zygosaccharomyces bailii is considered the most problematic acidic food spoilage yeast species due to its exceptional capacity to tolerate high concentrations of weak acids used as fungistatic preservatives at low pH. However, the mechanisms underlying its intrinsic remarkable tolerance to weak acids remain poorly understood. The identification of genes and mechanisms involved in Z. bailii acetic acid tolerance was on the focus of this study. For this, a genomic library from the highly acetic acid tolerant hybrid strain ISA1307, derived from Z. bailii and a closely related species and isolated from a sparkling wine production plant, was screened for acetic acid tolerance genes. This screen was based on the transformation of an acetic acid susceptible Saccharomyces cerevisiae mutant deleted for the gene encoding the acetic acid resistance determinant transcription factor Haa1. RESULTS: The expression of 31 different DNA inserts from ISA1307 strain genome was found to significantly increase the host cell tolerance to acetic acid. The in silico analysis of these inserts was facilitated by the recently available genome sequence of this strain. In total, 65 complete or truncated ORFs were identified as putative determinants of acetic acid tolerance and an S. cerevisiae gene homologous to most of them was found. These include genes involved in cellular transport and transport routes, protein fate, protein synthesis, amino acid metabolism and transcription. The role of strong candidates in Z. bailii and S. cerevisiae acetic acid tolerance was confirmed based on homologous and heterologous expression analyses. CONCLUSIONS: ISA1307 genes homologous to S. cerevisiae genes GYP8, WSC4, PMT1, KTR7, RKR1, TIF3, ILV3 and MSN4 are proposed as strong candidate determinants of acetic acid tolerance. The ORF ZBAI_02295 that contains a functional domain associated to the uncharacterised integral membrane proteins of unknown function of the DUP family is also suggested as a relevant tolerance determinant. The genes ZbMSN4 and ZbTIF3, encoding a putative stress response transcription factor and a putative translation initiation factor, were confirmed as determinants of acetic acid tolerance in both Z. bailii and S. cerevisiae. This study provides valuable indications on the cellular components, pathways and processes to be targeted in order to control food spoilage by the highly acetic acid tolerant Z. bailii and Z. bailii-derived strains. Additionally, this information is essential to guide the improvement of yeast cells robustness against acetic acid if the objective is their use as cell factories.

Novel, Synergistic Antifungal Combinations that Target Translation Fidelity.

There is an unmet need for new antifungal or fungicide treatments, as resistance to existing treatments grows. Combination treatments help to combat resistance. Here we develop a novel, effective target for combination antifungal therapy. Different aminoglycoside antibiotics combined with different sulphate-transport inhibitors produced strong, synergistic growth-inhibition of several fungi. Combinations decreased the respective MICs by ≥8-fold. Synergy was suppressed in yeast mutants resistant to effects of sulphate-mimetics (like chromate or molybdate) on sulphate transport. By different mechanisms, aminoglycosides and inhibition of sulphate transport cause errors in mRNA translation. The mistranslation rate was stimulated up to 10-fold when the agents were used in combination, consistent with this being the mode of synergistic action. A range of undesirable fungi were susceptible to synergistic inhibition by the combinations, including the human pathogens Candida albicans, C. glabrata and Cryptococcus neoformans, the food spoilage organism Zygosaccharomyces bailii and the phytopathogens Rhizoctonia solani and Zymoseptoria tritici. There was some specificity as certain fungi were unaffected. There was no synergy against bacterial or mammalian cells. The results indicate that translation fidelity is a promising new target for combinatorial treatment of undesirable fungi, the combinations requiring substantially decreased doses of active components compared to each agent alone.