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Many cancer cells share with yeast a preference for fermentation over respiration, which is associated with overactive glucose uptake and breakdown, a phenomenon called the Warburg effect in cancer cells. The yeast tps1Δ mutant shows even more pronounced hyperactive glucose uptake and phosphorylation causing glycolysis to stall at GAPDH, initiation of apoptosis through overactivation of Ras and absence of growth on glucose. The goal of the present work was to use the yeast tps1Δ strain to screen for novel compounds that would preferentially inhibit overactive glucose influx into glycolysis, while maintaining basal glucose catabolism. This is based on the assumption that the overactive glucose catabolism of the tps1Δ strain might have a similar molecular cause as the Warburg effect in cancer cells. We have isolated Warbicin® A as a compound restoring growth on glucose of the yeast tps1Δ mutant, showed that it inhibits the proliferation of cancer cells and isolated structural analogs by screening directly for cancer cell inhibition. The Warbicin® compounds are the first drugs that inhibit glucose uptake by both yeast Hxt and mammalian GLUT carriers. Specific concentrations did not evoke any major toxicity in mice but increase the amount of adipose tissue likely due to reduced systemic glucose uptake. Surprisingly, Warbicin® A inhibition of yeast sugar uptake depends on sugar phosphorylation, suggesting transport-associated phosphorylation as a target. In vivo and in vitro evidence confirms physical interaction between yeast Hxt7 and hexokinase. We suggest that reversible transport-associated phosphorylation by hexokinase controls the rate of glucose uptake through hydrolysis of the inhibitory ATP molecule in the cytosolic domain of glucose carriers and that in yeast tps1Δ cells and cancer cells reversibility is compromised, causing constitutively hyperactive glucose uptake and phosphorylation. Based on their chemical structure and properties, we suggest that Warbicin® compounds replace the inhibitory ATP molecule in the cytosolic domain of the glucose carriers, preventing hexokinase to cause hyperactive glucose uptake and catabolism.

Acetate-producing Saccharomyces cerevisiae var. boulardii strains could exert improved effects on ulcerative colitis, which here, was preclinically evaluated in an acute dextran sodium sulphate induced model of colitis. Nine-week-old female mice were divided into 12 groups, receiving either drinking water or 2.75% dextran sodium sulphate for 7 days, combined with a daily gavage of various treatments with different levels of acetate accumulation: sham control (phosphate buffered saline, no acetate), non-probiotic control (Baker’s yeast, no acetate), probiotic control (Enterol®, transient acetate), and additionally several Saccharomyces cerevisiae var. boulardii strains with respectively no, high, and extra-high acetate accumulation. Disease activity was monitored daily, and feces samples were collected at different timepoints. On day 14, the mice were sacrificed, upon which blood and colonic tissue were collected for analysis. Disease activity in inflamed mice was lower when treated with the high-acetate-producing strain compared to sham and non-probiotic controls. The non-acetate-producing strain showed higher disease activity compared to the acetate-producing strains. Accordingly, higher histologic inflammation was observed in non- or transient-acetate-producing strains compared to the sham control, whereas this increase was not observed for high- and extra-high-acetate-producing strains upon induction of inflammation. These anti-inflammatory findings were confirmed by transcriptomic analysis of differentially expressed genes. Moreover, only the strain with the highest acetate production was superior in maintaining a stable gut microbial alpha-diversity upon inflammation. These findings support new possibilities for acetate-mediated management of inflammation in inflammatory bowel disease by administrating high-acetate-producing Saccharomyces cerevisae var. boulardii strains.

The organic fraction of municipal solid waste (OFMSW) and refuse-derived fuel (RDF) mainly consisting of paper/cardboard can be used as feedstock for the production of cellulosic ethanol. In this paper, an efficient technology is described to convert waste paper/cardboard into cellulosic ethanol. The process involves separation of the OF from the other components in the waste stream. An acid pretreatment is used to liberate the cellulosic fibers and the accessibility of the enzyme Cellic CTEC3 loading 3.75–11.25 FPU/g paper in a fed-batch addition up to 22.5% solid yield, 15 g sugars/l with a saccharification yield up to 90%. A semi-simultaneous fermentation process (SSFP) with a saccharomyces cerevisae strain MDS130 capable of fermenting both pentoses and hexoses are growing an ethanol titer (%v/v) of 8.4% on pilon-plant scale.

Abstract As part of the transition from a fossil resources-based economy to a bio-based economy, the production of platform chemicals by microbial cell factories has gained strong interest. 2,3-butanediol (2,3-BDO) has various industrial applications, but its production by microbial fermentation poses multiple challenges. We have engineered the bacterial 2,3-BDO synthesis pathway, composed of AlsS, AlsD and BdhA, in a pdc-negative version of an industrial Saccharomyces cerevisiae yeast strain. The high concentration of glycerol caused by the excess NADH produced in the pathway from glucose to 2,3-BDO was eliminated by overexpression of NoxE and also in a novel way by combined overexpression of NDE1, encoding mitochondrial external NADH dehydrogenase, and AOX1, encoding a heterologous alternative oxidase expressed inside the mitochondria. This was combined with strong downregulation of GPD1 and deletion of GPD2, to minimize glycerol production while maintaining osmotolerance. The HGS50 strain produced a 2,3-BDO titer of 121.04 g/L from 250 g/L glucose, the highest ever reported in batch fermentation, with a productivity of 1.57 g/L.h (0.08 g/L.h per gCDW) and a yield of 0.48 g/g glucose or with 96% the closest to the maximum theoretical yield ever reported. Expression of Lactococcus lactis NoxE, encoding a water-forming NADH oxidase, combined with similar genetic modifications, as well as expression of Candida albicans STL1, also minimized glycerol production while maintaining high osmotolerance. The HGS37 strain produced 130.64 g/L 2,3-BDO from 280 g/L glucose, with productivity of 1.58 g/L.h (0.11 g/L.h per gCDW). Both strains reach combined performance criteria adequate for industrial implementation.

The presence of sugar in the gut causes induction of SGLT1, the sodium/glucose cotransporter in intestinal epithelial cells (enterocytes), and this is accompanied by stimulation of sugar absorption. Sugar sensing was suggested to involve a G-protein coupled receptor and cAMP - protein kinase A signalling, but the sugar receptor has remained unknown. We show strong expression and co-localization with SGLT1 of the β2-adrenergic receptor (β2-AR) at the enterocyte apical membrane and reveal its role in stimulating glucose uptake from the gut by the sodium/glucose-linked transporter, SGLT1. Upon heterologous expression in different reporter systems, the β2-AR responds to multiple sugars in the mM range, consistent with estimated gut sugar levels after a meal. Most adrenergic receptor antagonists inhibit sugar signaling, while some differentially inhibit epinephrine and sugar responses. However, sugars did not inhibit binding of I125-cyanopindolol, a β2-AR antagonist, to the ligand-binding site in cell-free membrane preparations. This suggests different but interdependent binding sites. Glucose uptake into everted sacs from rat intestine was stimulated by epinephrine and sugars in a β2-AR-dependent manner. STD-NMR confirmed direct physical binding of glucose to the β2-AR. Oral administration of glucose with a non-bioavailable β2-AR antagonist lowered the subsequent increase in blood glucose levels, confirming a role for enterocyte apical β2-ARs in stimulating gut glucose uptake, and suggesting enterocyte β2-AR as novel drug target in diabetic and obese patients. Future work will have to reveal how glucose sensing by enterocytes and neuroendocrine cells is connected, and whether β2-ARs mediate glucose sensing also in other tissues.

Abstract Background The current shift from a fossil-resource based economy to a more sustainable, bio-based economy requires development of alternative production routes based on utilization of biomass for the many chemicals that are currently produced from petroleum. Muconic acid is an attractive platform chemical for the bio-based economy because it can be converted in chemicals with wide industrial applicability, such as adipic and terephthalic acid, and because its two double bonds offer great versatility for chemical modification. Results We have constructed a yeast cell factory converting glucose and xylose into muconic acid without formation of ethanol. We consecutively eliminated feedback inhibition in the shikimate pathway, inserted the heterologous pathway for muconic acid biosynthesis from 3-dehydroshikimate (DHS) by co-expression of DHS dehydratase from P. anserina, protocatechuic acid (PCA) decarboxylase (PCAD) from K. pneumoniae and oxygen-consuming catechol 1,2-dioxygenase (CDO) from C. albicans, eliminated ethanol production by deletion of the three PDC genes and minimized PCA production by enhancing PCAD overexpression and production of its co-factor. The yeast pitching rate was increased to lower high biomass formation caused by the compulsory aerobic conditions. Maximal titers of 4 g/L, 4.5 g/L and 3.8 g/L muconic acid were reached with glucose, xylose, and a mixture, respectively. The use of an elevated initial sugar level, resulting in muconic acid titers above 2.5 g/L, caused stuck fermentations with incomplete utilization of the sugar. Application of polypropylene glycol 4000 (PPG) as solvent for in situ product removal during the fermentation shows that this is not due to toxicity by the muconic acid produced. Conclusions This work has developed an industrial yeast strain able to produce muconic acid from glucose and also with great efficiency from xylose, without any ethanol production, minimal production of PCA and reaching the highest titers in batch fermentation reported up to now. Utilization of higher sugar levels remained conspicuously incomplete. Since this was not due to product inhibition by muconic acid or to loss of viability, an unknown, possibly metabolic bottleneck apparently arises during muconic acid fermentation with high sugar levels and blocks further sugar utilization.

Abstract Background Presence of inhibitory chemicals in lignocellulose hydrolysates is a major hurdle for production of second-generation bioethanol. Especially cheaper pre-treatment methods that ensure an economical viable production process generate high levels of these inhibitory chemicals. The effect of several of these inhibitors has been extensively studied with non-xylose-fermenting laboratory strains, in synthetic media, and usually as single inhibitors, or with inhibitor concentrations much higher than those found in lignocellulose hydrolysates. However, the relevance of individual inhibitors in inhibitor-rich lignocellulose hydrolysates has remained unclear. Results The relative importance for inhibition of ethanol fermentation by two industrial second-generation yeast strains in five lignocellulose hydrolysates, from bagasse, corn cobs and spruce, has now been investigated by spiking higher concentrations of each compound in a concentration range relevant for industrial hydrolysates. The strongest inhibition was observed with industrially relevant concentrations of furfural causing partial inhibition of both D-glucose and D-xylose consumption. Addition of 3 or 6 g/L furfural strongly reduced the ethanol titer obtained with strain MD4 in all hydrolysates evaluated, in a range of 34 to 51% and of 77 to 86%, respectively. This was followed by 5-hydroxymethylfurfural, acetic acid and formic acid, for which in general, industrially relevant concentrations caused partial inhibition of D-xylose fermentation. On the other hand, spiking with levulinic acid, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid or vanillin caused little inhibition compared to unspiked hydrolysate. The further evolved MD4 strain generally showed superior performance compared to the previously developed strain GSE16-T18. Conclusion The results highlight the importance of individual inhibitor evaluation in a medium containing a genuine mix of inhibitors as well as the ethanol that is produced by the fermentation. They also highlight the potential of increasing yeast inhibitor tolerance for improving industrial process economics.

One of the major bottlenecks in lactic acid production using microbial fermentation is the detrimental influence lactic acid accumulation poses on the lactic acid producing cells. The accumulation of lactic acid results in many negative effects on the cell such as intracellular acidification, anion accumulation, membrane perturbation, disturbed amino acid trafficking, increased turgor pressure, ATP depletion, ROS accumulation, metabolic dysregulation and metal chelation. In this review, the manner in which Saccharomyces cerevisiae deals with these issues will be discussed extensively not only for lactic acid as a singular stress factor but also in combination with other stresses. In addition, different methods to improve lactic acid tolerance in S. cerevisiae using targeted and non-targeted engineering methods will be discussed.

A major signal transduction pathway regulating cell growth and many associated physiological properties as a function of nutrient availability in the yeast Saccharomyces cerevisiae is the protein kinase A (PKA) pathway. Glucose activation of PKA is mediated by G-protein coupled receptor (GPCR) Gpr1, and secondary messenger cAMP. Other nutrients, including nitrogen, phosphate and sulfate, activate PKA in accordingly-starved cells through nutrient transceptors, but apparently without cAMP signaling. We have now used an optimized EPAC-based fluorescence resonance energy transfer (FRET) sensor to precisely monitor in vivo cAMP levels after nutrient addition. We show that GPCR-mediated glucose activation of PKA is correlated with a rapid transient increase in the cAMP level in vivo, whereas nutrient transceptor-mediated activation by nitrogen, phosphate or sulfate, is not associated with any significant increase in cAMP in vivo. We also demonstrate direct physical interaction between the Gap1 amino acid transceptor and the catalytic subunits of PKA, Tpk1, 2 and 3. In addition, we reveal a conserved consensus motif in the nutrient transceptors that is also present in Bcy1, the regulatory subunit of PKA. This suggests that nutrient transceptor activation of PKA may be mediated by direct release of bound PKA catalytic subunits, triggered by the conformational changes occurring during transport of the substrate by the transceptor. Our results support a model in which nutrient transceptors are evolutionary ancestors of GPCRs, employing a more primitive direct signaling mechanism compared to the indirect cAMP second-messenger signaling mechanism used by GPCRs for activation of PKA.

Abstract Background High acetic acid tolerance is of major importance in industrial yeast strains used for second-generation bioethanol production, because of the high acetic acid content of lignocellulose hydrolysates. It is also important in first-generation starch hydrolysates and in sourdoughs containing significant acetic acid levels. We have previously identified snf4 E269* as a causative allele in strain MS164 obtained after whole-genome (WG) transformation and selection for improved acetic acid tolerance. Results We have now performed polygenic analysis with the same WG transformant MS164 to identify novel causative alleles interacting with snf4 E269* to further enhance acetic acid tolerance, from a range of 0.8–1.2% acetic acid at pH 4.7, to previously unmatched levels for Saccharomyces cerevisiae. For that purpose, we crossed the WG transformant with strain 16D, a previously identified strain displaying very high acetic acid tolerance. Quantitative trait locus (QTL) mapping with pooled-segregant whole-genome sequence analysis identified four major and two minor QTLs. In addition to confirmation of snf4 E269* in QTL1, we identified six other genes linked to very high acetic acid tolerance, TRT2, MET4, IRA2 and RTG1 and a combination of MSH2 and HAL9, some of which have never been connected previously to acetic acid tolerance. Several of these genes appear to be wild-type alleles that complement defective alleles present in the other parent strain. Conclusions The presence of several novel causative genes highlights the distinct genetic basis and the strong genetic background dependency of very high acetic acid tolerance. Our results suggest that elimination of inferior mutant alleles might be equally important for reaching very high acetic acid tolerance as introduction of rare superior alleles. The superior alleles of MET4 and RTG1 might be useful for further improvement of acetic acid tolerance in specific industrial yeast strains.

Abstract Background The search for sustainable energy sources has become a worldwide issue, making the development of efficient biofuel production processes a priority. Immobilization of second-generation (2G) xylose-fermenting Saccharomyces cerevisiae strains is a promising approach to achieve economic viability of 2G bioethanol production from undetoxified hydrolysates through operation at high cell load and mitigation of inhibitor toxicity. In addition, the use of a fixed-bed reactor can contribute to establish an efficient process because of its distinct advantages, such as high conversion rate per weight of biocatalyst and reuse of biocatalyst. Results This work assessed the influence of alginate entrapment on the tolerance of recombinant S. cerevisiae to acetic acid. Encapsulated GSE16-T18SI.1 (T18) yeast showed an outstanding performance in repeated batch fermentations with cell recycling in YPX medium supplemented with 8 g/L acetic acid (pH 5.2), achieving 10 cycles without significant loss of productivity. In the fixed-bed bioreactor, a high xylose fermentation rate with ethanol yield and productivity values of 0.38 gethanol/gsugars and 5.7 g/L/h, respectively were achieved in fermentations using undetoxified sugarcane bagasse hemicellulose hydrolysate, with and without medium recirculation. Conclusions The performance of recombinant strains developed for 2G ethanol production can be boosted strongly by cell immobilization in alginate gels. Yeast encapsulation allows conducting fermentations in repeated batch mode in fixed-bed bioreactors with high xylose assimilation rate and high ethanol productivity using undetoxified hemicellulose hydrolysate.

Natural yeast with superior fermentative traits can serve as a platform for the development of recombinant strains that can be used to improve the sustainability of bioethanol production from starch. This process will benefit from a consolidated bioprocessing (CBP) approach where an engineered strain producing amylases directly converts starch into ethanol. The yeast Saccharomyces cerevisiae L20, previously selected as outperforming the benchmark yeast Ethanol Red, was here subjected to a comparative genomic investigation using a dataset of industrial S. cerevisiae strains. Along with Ethanol Red, strain L20 was then engineered for the expression of α-amylase amyA and glucoamylase glaA genes from Aspergillus tubingensis by employing two different approaches (delta integration and CRISPR/Cas9). A correlation between the number of integrated copies and the hydrolytic abilities of the recombinants was investigated. L20 demonstrated important traits for the construction of a proficient CBP yeast. Despite showing a close relatedness to commercial wine yeast and the benchmark Ethanol Red, a unique profile of gene copy number variations (CNVs) was found in L20, mainly encoding membrane transporters and secretion pathway proteins but also the fermentative metabolism. Moreover, the genome annotation disclosed seven open reading frames (ORFs) in L20 that are absent in the reference S288C genome. Genome engineering was successfully implemented for amylase production. However, with equal amylase gene copies, L20 proved its proficiency as a good enzyme secretor by exhibiting a markedly higher amylolytic activity than Ethanol Red, in compliance to the findings of the genomic exploration. The recombinant L20 dT8 exhibited the highest amylolytic activity and produced more than 4 g/L of ethanol from 2% starch in a CBP setting without the addition of supplementary enzymes. Based on the performance of this strain, an amylase/glucoamylase ratio of 1:2.5 was suggested as baseline for further improvement of the CBP ability. Overall, L20 showed important traits for the future construction of a proficient CBP yeast. As such, this work shows that natural S. cerevisiae strains can be used for the expression of foreign secreted enzymes, paving the way to strain improvement for the starch-to-bioethanol route.

Short-chain fatty acids as well as their bacterial producers are of increasing interest in inflammatory bowel diseases. Although less studied compared to butyrate, acetate might also be of interest as it may be less toxic to epithelial cells, stimulate butyrate-producing bacteria by cross-feeding, and have anti-inflammatory and barrier-protective properties. Moreover, one of the causative factors of the probiotic potency of Saccharomyces cerevisae var. boulardii is thought to be its high acetate production. Therefore, the objective was to preclinically assess the effects of high acetate concentrations on inflammation and barrier integrity in organoid-based monolayer cultures from ulcerative colitis patients. Confluent organoid-derived colonic epithelial monolayers (n = 10) were exposed to basolateral inflammatory stimulation or control medium. After 24 h, high acetate or control medium was administered apically for an additional 48 h. Changes in TEER were measured after 48 h. Expression levels of barrier genes and inflammatory markers were determined by qPCR. Pro-inflammatory proteins in the supernatant were quantified using the MSD platform. Increased epithelial resistance was observed with high acetate administration in both inflamed and non-inflamed conditions, together with decreased expression levels of IL8 and TNFα and CLDN1. Upon high acetate administration to inflamed monolayers, upregulation of HIF1α, MUC2, and MKI67, and a decrease of the majority of pro-inflammatory cytokines was observed. In our patient-derived human epithelial cell culture model, a protective effect of high acetate administration on epithelial resistance, barrier gene expression, and inflammatory protein production was observed. These findings open up new possibilities for acetate-mediated management of barrier defects and inflammation in IBD.

Abstract Background Xylose isomerase (XI) and xylose reductase/xylitol dehydrogenase (XR/XDH) pathways have been extensively used to confer xylose assimilation capacity to Saccharomyces cerevisiae and tackle one of the major bottlenecks in the attainment of economically viable lignocellulosic ethanol production. Nevertheless, there is a lack of studies comparing the efficiency of those pathways both separately and combined. In this work, the XI and/or XR/XDH pathways were introduced into two robust industrial S. cerevisiae strains, evaluated in synthetic media and corn cob hemicellulosic hydrolysate and the results were correlated with the differential enzyme activities found in the xylose-pathway engineered strains. Results The sole expression of XI was found to increase the fermentative capacity of both strains in synthetic media at 30 °C and 40 °C: decreasing xylitol accumulation and improving xylose consumption and ethanol production. Similar results were observed in fermentations of detoxified hydrolysate. However, in the presence of lignocellulosic-derived inhibitors, a positive synergistic effect resulted from the expression of both XI and XR/XDH, possibly caused by a cofactor equilibrium between the XDH and furan detoxifying enzymes, increasing the ethanol yield by more than 38%. Conclusions This study clearly shows an advantage of using the XI from Clostridium phytofermentans to attain high ethanol productivities and yields from xylose. Furthermore, and for the first time, the simultaneous utilization of XR/XDH and XI pathways was compared to the single expression of XR/XDH or XI and was found to improve ethanol production from non-detoxified hemicellulosic hydrolysates. These results extend the knowledge regarding S. cerevisiae xylose assimilation metabolism and pave the way for the construction of more efficient strains for use in lignocellulosic industrial processes.

The development of biorefineries brings the necessity of an efficient consumption of all sugars released from biomasses, including xylose. In addition, the presence of inhibitors in biomass hydrolysates is one of the main challenges in bioprocess feasibility. In this study, the application of Ca-alginate hybrid gels in the immobilization of xylose-consuming recombinant yeast was explored with the aim of improving the tolerance of inhibitors. The recombinant yeast Saccharomyces cerevisiae GSE16-T18SI.1 (T18) was immobilized in Ca-alginate and Ca-alginate–chitosan hybrid beads, and its performance on xylose fermentation was evaluated in terms of tolerance to different acetic acid concentrations (0–12 g/L) and repeated batches of crude sugarcane bagasse hemicellulose hydrolysate. The use of the hybrid gel improved yeast performance in the presence of 12 g/L of acetic acid, achieving 1.13 g/L/h of productivity and reaching 75% of the theoretical ethanol yield, with an improvement of 32% in the xylose consumption rate (1:1 Vbeads/Vmedium, 35 °C, 150 rpm and pH 5.2). The use of hybrid alginate–chitosan gel also led to better yeast performance at crude hydrolysate, yielding one more batch than the pure-alginate beads. These results demonstrate the potential of a hybrid gel as an approach that could increase 2G ethanol productivity and allow cell recycling for a longer period.

In this work, we have identified for the first time the causative elements in a eukaryotic organism introduced by applying whole-genome transformation and responsible for the selectable trait of interest, i.e., high temperature tolerance. Surprisingly, the whole-genome transformants contained just a few single nucleotide polymorphisms (SNPs), which were unrelated to the sequence of the donor DNA.

ABSTRACT Whereas the yeast Saccharomyces cerevisiae shows great preference for glucose as a carbon source, a deletion mutant in trehalose-6-phosphate synthase, tps1Δ, is highly sensitive to even a few millimolar glucose, which triggers apoptosis and cell death. Glucose addition to tps1Δ cells causes deregulation of glycolysis with hyperaccumulation of metabolites upstream and depletion downstream of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The apparent metabolic barrier at the level of GAPDH has been difficult to explain. We show that GAPDH isozyme deletion, especially Tdh3, further aggravates glucose sensitivity and metabolic deregulation of tps1Δ cells, but overexpression does not rescue glucose sensitivity. GAPDH has an unusually high pH optimum of 8.0 to 8.5, which is not altered by tps1Δ. Whereas glucose causes short, transient intracellular acidification in wild-type cells, in tps1Δ cells, it causes permanent intracellular acidification. The hxk2Δ and snf1Δ suppressors of tps1Δ restore the transient acidification. These results suggest that GAPDH activity in the tps1Δ mutant may be compromised by the persistently low intracellular pH. Addition of NH4Cl together with glucose at high extracellular pH to tps1Δ cells abolishes the pH drop and reduces glucose-6-phosphate (Glu6P) and fructose-1,6-bisphosphate (Fru1,6bisP) hyperaccumulation. It also reduces the glucose uptake rate, but a similar reduction in glucose uptake rate in a tps1Δ hxt2,4,5,6,7Δ strain does not prevent glucose sensitivity and Fru1,6bisP hyperaccumulation. Hence, our results suggest that the glucose-induced intracellular acidification in tps1Δ cells may explain, at least in part, the apparent glycolytic bottleneck at GAPDH but does not appear to fully explain the extreme glucose sensitivity of the tps1Δ mutant. IMPORTANCE Glucose catabolism is the backbone of metabolism in most organisms. In spite of numerous studies and extensive knowledge, major controls on glycolysis and its connections to the other metabolic pathways remain to be discovered. A striking example is provided by the extreme glucose sensitivity of the yeast tps1Δ mutant, which undergoes apoptosis in the presence of just a few millimolar glucose. Previous work has shown that the conspicuous glucose-induced hyperaccumulation of the glycolytic metabolite fructose-1,6-bisphosphate (Fru1,6bisP) in tps1Δ cells triggers apoptosis through activation of the Ras-cAMP-protein kinase A (PKA) signaling pathway. However, the molecular cause of this Fru1,6bisP hyperaccumulation has remained unclear. We now provide evidence that the persistent drop in intracellular pH upon glucose addition to tps1Δ cells likely compromises the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a major glycolytic enzyme downstream of Fru1,6bisP, due to its unusually high pH optimum. Our work highlights the potential importance of intracellular pH fluctuations for control of major metabolic pathways.

Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cellular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the definition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death routines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the authors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the progress of this vibrant field of research.

Yeast and cancer cells both favor sugar fermentation in aerobic conditions. Here the authors describe a conserved mechanism from yeast to mammals where the glycolysis intermediate fructose-1,6-bisphosphate binds Cdc25/Sos1 and couples increased glycolytic flux to increased Ras proto-oncoprotein activity.

Abstract Background Non-conventional yeasts present a huge, yet barely exploited, resource of yeast biodiversity for industrial applications. This presents a great opportunity to explore alternative ethanol-fermenting yeasts that are more adapted to some of the stress factors present in the harsh environmental conditions in second-generation (2G) bioethanol fermentation. Extremely tolerant yeast species are interesting candidates to investigate the underlying tolerance mechanisms and to identify genes that when transferred to existing industrial strains could help to design more stress-tolerant cell factories. For this purpose, we performed a high-throughput phenotypic evaluation of a large collection of non-conventional yeast species to identify the tolerance limits of the different yeast species for desirable stress tolerance traits in 2G bioethanol production. Next, 12 multi-tolerant strains were selected and used in fermentations under different stressful conditions. Five strains out of which, showing desirable fermentation characteristics, were then evaluated in small-scale, semi-anaerobic fermentations with lignocellulose hydrolysates. Results Our results revealed the phenotypic landscape of many non-conventional yeast species which have not been previously characterized for tolerance to stress conditions relevant for bioethanol production. This has identified for each stress condition evaluated several extremely tolerant non-Saccharomyces yeasts. It also revealed multi-tolerance in several yeast species, which makes those species good candidates to investigate the molecular basis of a robust general stress tolerance. The results showed that some non-conventional yeast species have similar or even better fermentation efficiency compared to S. cerevisiae in the presence of certain stressful conditions. Conclusion Prior to this study, our knowledge on extreme stress-tolerant phenotypes in non-conventional yeasts was limited to only few species. Our work has now revealed in a systematic way the potential of non-Saccharomyces species to emerge either as alternative host species or as a source of valuable genetic information for construction of more robust industrial S. serevisiae bioethanol production yeasts. Striking examples include yeast species like Pichia kudriavzevii and Wickerhamomyces anomalus that show very high tolerance to diverse stress factors. This large-scale phenotypic analysis has yielded a detailed database useful as a resource for future studies to understand and benefit from the molecular mechanisms underlying the extreme phenotypes of non-conventional yeast species.

Multiple types of nutrient transceptors, membrane proteins that combine a transporter and receptor function, have now been established in a variety of organisms. However, so far all established transceptors utilize one of the macronutrients, glucose, amino acids, ammonium, nitrate, phosphate or sulfate, as substrate. This is also true for the Saccharomyces cerevisiae transceptors mediating activation of the PKA pathway upon re-addition of a macronutrient to glucose-repressed cells starved for that nutrient, re-establishing a fermentable growth medium. We now show that the yeast high-affinity iron transporter Ftr1 and high-affinity zinc transporter Zrt1 function as transceptors for the micronutrients iron and zinc. We show that replenishment of iron to iron-starved cells or zinc to zinc-starved cells triggers within 1-2 minutes a rapid surge in trehalase activity, a well-established PKA target. The activation with iron is dependent on Ftr1 and with zinc on Zrt1, and we show that it is independent of intracellular iron and zinc levels. Similar to the transceptors for macronutrients, Ftr1 and Zrt1 are strongly induced upon iron and zinc starvation, respectively, and they are rapidly downregulated by substrate-induced endocytosis. Our results suggest that transceptor-mediated signaling to the PKA pathway may occur in all cases where glucose-repressed yeast cells have been starved first for an essential nutrient, causing arrest of growth and low activity of the PKA pathway, and subsequently replenished with the lacking nutrient to re-establish a fermentable growth medium. The broadness of the phenomenon also makes it likely that nutrient transceptors use a common mechanism for signaling to the PKA pathway.

Sulfonates are a major source of sulphur for soil microbes but their cellular uptake is still not fully understood. Here the authors show thatSaccharomyces cerevisiae YIL166C(SOA1)encodes for an inorganic sulphur transporter that can also function as a sulfonate and choline sulphate transporter.

Isolation of mutants in populations of microorganisms has been a valuable tool in experimental genetics for decades. The main disadvantage, however, is the inability of isolating mutants in non-selectable polygenic traits. Most traits of organisms, however, are non-selectable and polygenic, including industrially important properties of microorganisms. The advent of powerful technologies for polygenic analysis of complex traits has allowed simultaneous identification of multiple causative mutations among many thousands of irrelevant mutations. We now show that this also applies to haploid strains of which the genome has been loaded with induced mutations so as to affect as many non-selectable, polygenic traits as possible. We have introduced about 900 mutations into single haploid yeast strains using multiple rounds of EMS mutagenesis, while maintaining the mating capacity required for genetic mapping. We screened the strains for defects in flavor production, an important non-selectable, polygenic trait in yeast alcoholic beverage production. A haploid strain with multiple induced mutations showing reduced ethyl acetate production in semi-anaerobic fermentation, was selected and the underlying quantitative trait loci (QTLs) were mapped using pooled-segregant whole-genome sequence analysis after crossing with an unrelated haploid strain. Reciprocal hemizygosity analysis and allele exchange identified PMA1 and CEM1 as causative mutant alleles and TPS1 as a causative genetic background allele. The case of CEM1 revealed that relevant mutations without observable effect in the haploid strain with multiple induced mutations (in this case due to defective mitochondria) can be identified by polygenic analysis as long as the mutations have an effect in part of the segregants (in this case those that regained fully functional mitochondria). Our results show that genomic saturation mutagenesis combined with complex trait polygenic analysis could be used successfully to identify causative alleles underlying many non-selectable, polygenic traits in small collections of haploid strains with multiple induced mutations.

ABSTRACT Flavor production in yeast fermentation is of paramount importance for industrial production of alcoholic beverages. Although major enzymes of flavor compound biosynthesis have been identified, few specific mutations responsible for strain diversity in flavor production are known. The ATF1-encoded alcohol acetyl coenzyme A (acetyl-CoA) transferase (AATase) is responsible for the majority of acetate ester biosynthesis, but other components affecting strain diversity remain unknown. We have performed parallel polygenic analysis of low production of ethyl acetate, a compound with an undesirable solvent-like off-flavor, in strains with and without deletion of ATF1. We identified two unique causative mutations, eat1K179fs and snf8E148*, not present in any other sequenced yeast strain and responsible for most ethyl acetate produced in absence of ATF1. EAT1 encodes a putative mitochondrial ethanol acetyl-CoA transferase (EATase) and its overexpression, but not that of EAT1K179fs, and strongly increases ethyl acetate without affecting other flavor acetate esters. Unexpectedly, a higher level of acetate esters (including ethyl acetate) was produced when eat1K179fs was present together with ATF1 in the same strain, suggesting that the Eat1 and Atf1 enzymes are intertwined. On the other hand, introduction of snf8E148* lowered ethyl acetate levels also in the presence of ATF1, and it affected other aroma compounds, growth, and fermentation as well. Engineering of snf8E148* in three industrial yeast strains (for production of wine, saké, and ale beer) and fermentation in an application-relevant medium showed a high but strain-dependent potential for flavor enhancement. Our work has identified EAT1 and SNF8 as new genetic elements determining ethyl acetate production diversity in yeast strains. IMPORTANCE Basic research with laboratory strains of the yeast Saccharomyces cerevisiae has identified the structural genes of most metabolic enzymes, as well as genes encoding major regulators of metabolism. On the other hand, more recent work on polygenic analysis of yeast biodiversity in natural and industrial yeast strains is revealing novel components of yeast metabolism. A major example is the metabolism of flavor compounds, a particularly important property of industrial yeast strains used for the production of alcoholic beverages. In this work, we have performed polygenic analysis of production of ethyl acetate, an important off-flavor compound in beer and other alcoholic beverages. To increase the chances of identifying novel components, we have used in parallel a wild-type strain and a strain with a deletion of ATF1 encoding the main enzyme of acetate ester biosynthesis. This revealed a new structural gene, EAT1, encoding a putative mitochondrial enzyme, which was recently identified as an ethanol acetyl-CoA transferase in another yeast species. We also identified a novel regulatory gene, SNF8, which has not previously been linked to flavor production. Our results show that polygenic analysis of metabolic traits in the absence of major effector genes can reveal novel structural and regulatory genes. The mutant alleles identified can be used to affect the flavor profile in industrial yeast strains for production of alcoholic beverages in more subtle ways than by deletion or overexpression of the already known major effector genes and without significantly altering other industrially important traits. The effect of the novel variants was dependent on the genetic background, with a highly desirable outcome in the flavor profile of an ale brewing yeast.

The nutrient composition of the medium has dramatic effects on many cellular properties in the yeast Saccharomyces cerevisiae. In addition to the well-known specific responses to starvation for an essential nutrient, like nitrogen or phosphate, the presence of fermentable sugar or a respirative carbon source leads to predominance of fermentation or respiration, respectively. Fermenting and respiring cells also show strong differences in other properties, like storage carbohydrate levels, general stress tolerance and cellular growth rate. However, the main glucose repression pathway, which controls the switch between respiration and fermentation, is not involved in control of these properties. They are controlled by the protein kinase A (PKA) pathway. Addition of glucose to respiring yeast cells triggers cAMP synthesis, activation of PKA and rapid modification of its targets, like storage carbohydrate levels, general stress tolerance and growth rate. However, starvation of fermenting cells in a glucose medium for any essential macro- or micro-nutrient counteracts this effect, leading to downregulation of PKA and its targets concomitant with growth arrest and entrance into G0. Re-addition of the lacking nutrient triggers rapid activation of the PKA pathway, without involvement of cAMP as second messenger. Investigation of the sensing mechanism has revealed that the specific high-affinity nutrient transporter(s) induced during starvation function as transporter-receptors or transceptors for rapid activation of PKA upon re-addition of the missing substrate. In this way, transceptors have been identified for amino acids, ammonium, phosphate, sulfate, iron, and zinc. We propose a hypothesis for regulation of PKA activity by nutrient transceptors to serve as a conceptual framework for future experimentation. Many properties of transceptors appear to be similar to those of classical receptors and nutrient transceptors may constitute intermediate forms in the development of receptors from nutrient transporters during evolution. The nutrient-sensing transceptor system in yeast for activation of the PKA pathway has served as a paradigm for similar studies on candidate nutrient transceptors in other eukaryotes and we succinctly discuss the many examples of transceptors that have already been documented in other yeast species, filamentous fungi, plants, and animals, including the examples in human cells.

ABSTRACT Flavor compound metabolism is one of the last areas in metabolism where multiple genes encoding biosynthetic enzymes are still unknown. A major challenge is the involvement of side activities of enzymes having their main function in other areas of metabolism. We have applied pooled-segregant whole-genome sequence analysis to identify novel Saccharomyces cerevisiae genes affecting production of phenylethyl acetate (2-PEAc). This is a desirable flavor compound of major importance in alcoholic beverages imparting rose- and honey-like aromas, with production of high 2-PEAc levels considered a superior trait. Four quantitative trait loci (QTLs) responsible for high 2-PEAc production were identified, with two loci each showing linkage to the genomes of the BTC.1D and ER18 parents. The first two loci were investigated further. The causative genes were identified by reciprocal allele swapping into both parents using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9. The superior allele of the first major causative gene, FAS2, was dominant and contained two unique single nucleotide polymorphisms (SNPs) responsible for high 2-PEAc production that were not present in other sequenced yeast strains. FAS2 encodes the alpha subunit of the fatty acid synthetase complex. Surprisingly, the second causative gene was a mutant allele of TOR1, a gene involved in nitrogen regulation. Exchange of both superior alleles in the ER18 parent strain increased 2-PEAc production 70%, nearly to the same level as in the best superior segregant. Our results show that polygenic analysis combined with CRISPR/Cas9-mediated allele exchange is a powerful tool for identification of genes encoding missing metabolic enzymes and for development of industrial yeast strains generating novel flavor profiles in alcoholic beverages. IMPORTANCE Multiple reactions in flavor metabolism appear to be catalyzed by side activities of other enzymes that have been difficult to identify. We have applied genetic mapping of quantitative trait loci in the yeast Saccharomyces cerevisiae to identify mutant alleles of genes determining the production of phenylethyl acetate, an important flavor compound imparting rose- and honey-like aromas to alcoholic beverages. We identified a unique, dominant allele of FAS2 that supports high production of phenylethyl acetate. FAS2 encodes a subunit of the fatty acid synthetase complex and apparently exerts an important side activity on one or more alternative substrates in flavor compound synthesis. The second mutant allele contained a nonsense mutation in TOR1, a gene involved in nitrogen regulation of growth. Together the two alleles strongly increased the level of phenylethyl acetate. Our work highlights the potential of genetic mapping of quantitative phenotypic traits to identify novel enzymes and regulatory components in yeast metabolism, including regular metabolic enzymes with unknown side activities responsible for biosynthesis of specific flavor compounds. The superior alleles identified can be used to develop industrial yeast strains generating novel flavor profiles in alcoholic beverages.