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Sterols are essential components of the yeast membrane and their synthesis requires oxygen. Yet, Saccharomyces cerevisiae has developed the ability to take up sterols from the medium under anaerobiosis. Here we investigated sterol uptake efficiency and the expression of genes related to sterol import in Saccharomyces and non-Saccharomyces wine yeast species fermenting under anaerobic conditions. The sterol uptake efficiency of 39 strains was evaluated by flow cytometry (with 25-NBD Cholesterol, a fluorescent cholesterol probe introduced in the medium) and we found an important discrepancy between Saccharomyces and non-Saccharomyces wine yeast species that we correlated to a lower final cell population and a lower fermentation rate. A high uptake of sterol was observed in the various Saccharomyces strains. Spot tests performed on 13 of these strains confirmed the differences between Saccharomyces and non-Saccharomyces strains, suggesting that the presence of the sterol uptake transporters AUS1 and PDR11 could cause these discrepancies. Indeed, we could not find any homologue to these genes in the genome of Hanseniaspora uvarum, H. guillermondii, Lachancea thermotolerans, Torulaspora delbreueckii, Metschnikowia pulcherrima, or Starmarella bacillaris species. The specialization of sterol import function for post genome-duplication species may have favored growth under anaerobiosis.

The yeast Lachancea thermotolerans is of significant biotechnological interest, and selected strains of this species have become commonly used starter cultures in wine fermentation. However, the impact of this species on wine is frequently limited by the rapid dominance of Saccharomyces cerevisiae strains which are better adapted to wine alcoholic fermentation conditions. Previous studies have shown that the major limiting factor for L. thermotolerans competitive performance in the wine ecosystem is oxygen availability, and not ethanol levels as had been previously suggested. Here we investigated the transcriptional response of L. thermotolerans to anaerobiosis in wine fermentation conditions. The data show that L. thermotolerans broadly redirects gene expression towards genes involved in central carbon metabolism, lipid metabolism, remodeling of the cell wall as well as autophagy. Furthermore, the induction of genes that are likely involved in the generation of lactate indicates a redirection of metabolic flux towards this metabolite. The data provide the first insight into the oxygen-dependent response of L. thermotolerans and suggest potential genetic targets to improve lactate production and/or anaerobic fermentation performance of this yeast.

Kluyveromyces marxianus is a thermotolerant, ethanol-producing yeast that requires oxygen for efficient ethanol fermentation. Under anaerobic conditions, glucose consumption and ethanol production are retarded, suggesting that oxygen affects the metabolic state of K. marxianus. Mitochondria require oxygen to function, and their forms and number vary according to environmental conditions. In this study, the effect of anoxia on mitochondrial behavior in K. marxianus was examined. Under aerobic growth conditions, mitochondria-targeted GFP exhibited a tubular and dotted localization, representing a typical mitochondrial morphology, but under anaerobic conditions, GFP localized in vacuoles, suggesting that mitophagy occurs under anaerobic conditions. To confirm mitophagy induction, the ATG32, ATG8, ATG11 and ATG19 genes were disrupted. Vacuolar localization of mitochondria-targeted GFP under anaerobic conditions was interrupted in the Δatg32 and Δatg8 strains but not the Δatg11 and Δatg19 strains. Electron microscopy revealed mitochondria-like membrane components in the vacuoles of wild-type cells grown under anaerobic conditions. Quantitative analyses using mitochondria-targeted Pho8 demonstrated that mitophagy was induced in K. marxianus by anoxia but not nitrogen starvation. To the best of our knowledge, this is the first demonstration of anoxia-induced mitophagy in yeasts.

We sought to investigate how far the growth of Saccharomyces cerevisiae under full anaerobiosis is dependent on the widely used anaerobic growth factors (AGF) ergosterol and oleic acid. A continuous cultivation setup was employed and, even forcing ultrapure N2 gas through an O2 trap upstream of the bioreactor, neither cells from S. cerevisiae CEN.PK113–7D (a lab strain) nor from PE-2 (an industrial strain) washed out after an aerobic-to-anaerobic switch in the absence of AGF. S. cerevisiae PE-2 seemed to cope better than the laboratory strain with this extremely low O2 availability, since it presented higher biomass yield, lower specific rates of glucose consumption and CO2 formation, and higher survival at low pH. Lipid (fatty acid and sterol) composition dramatically altered when cells were grown anaerobically without AGF: saturated fatty acid, squalene and lanosterol contents increased, when compared to either cells grown aerobically or anaerobically with AGF. We concluded that these lipid alterations negatively affect cell viability during exposure to low pH or high ethanol titers.

Dekkera bruxellensis is considered a spoilage yeast in winemaking, brewing and fuel-ethanol production. However, there is growing evidence in the literature of its biotechnological potential. In this work, we surveyed 29 D. bruxellensis isolates from three countries and two different industrial origins (winemaking and fuel-ethanol production) for the metabolization of industrially relevant sugars. The isolates were characterized by the determination of their maximum specific growth rates, and by testing their ability to grow in the presence of 2-deoxy-d-glucose and antimycin A. Great diversity was observed among the isolates, with fuel-ethanol isolates showing overall higher specific growth rates than wine isolates. Preferences for galactose (three wine isolates) and for cellobiose or lactose (some fuel-ethanol isolates) were observed. Fuel-ethanol isolates were less sensitive than wine isolates to glucose catabolite repression (GCR) induction by 2-deoxy-d-glucose. In strictly anaerobic conditions, isolates selected for having high aerobic growth rates were able to ferment glucose, sucrose and cellobiose at fairly high rates without supplementation of casamino acids or yeast extract in the culture medium. The phenotypic diversity found among wine and fuel-ethanol isolates suggests adaptation to these environments. A possible application of some of the GCR-insensitive, fast-growing isolates in industrial processes requiring co-assimilation of different sugars is considered.

Glucose is the preferred nutrient for most living cells and is also a signaling molecule that modulates several cellular processes. Glucose regulates the expression of glucose permease genes in yeasts through signaling pathways dependent on plasma membrane glucose sensors. In the yeast Kluyveromyces lactis, sufficient levels of glucose induction of the low-affinity glucose transporter RAG1 gene also depends on a functional glycolysis, suggesting additional intracellular signaling. We have found that the expression of RAG1 gene is also induced by hypoxia in the presence of glucose, indicating that glucose and oxygen signaling pathways are interconnected. In this study we investigated the molecular mechanisms underlying this crosstalk. By analyzing RAG1 expression in various K. lactis mutants, we found that the bHLH transcriptional activator Sck1 is required for the hypoxic induction of RAG1 gene. The RAG1 promoter region essential for its hypoxic induction was identified by promoter deletion experiments. Taken together, these results show that the RAG1 glucose permease gene is synergistically induced by hypoxia and glucose and highlighted a novel role for the transcriptional activator Sck1 as a key mediator in this mechanism.

Expression of a heterologous xylose isomerase, deletion of the GRE3 aldose-reductase gene and overexpression of genes encoding xylulokinase (XKS1) and non-oxidative pentose-phosphate-pathway enzymes (RKI1, RPE1, TAL1, TKL1) enables aerobic growth of Saccharomyces cerevisiae on d-xylose. However, literature reports differ on whether anaerobic growth on d-xylose requires additional mutations. Here, CRISPR-Cas9-assisted reconstruction and physiological analysis confirmed an early report that this basic set of genetic modifications suffices to enable anaerobic growth on d-xylose in the CEN.PK genetic background. Strains that additionally carried overexpression cassettes for the transaldolase and transketolase paralogs NQM1 and TKL2 only exhibited anaerobic growth on d-xylose after a 7–10 day lag phase. This extended lag phase was eliminated by increasing inoculum concentrations from 0.02 to 0.2 g biomass L−1. Alternatively, a long lag phase could be prevented by sparging low-inoculum-density bioreactor cultures with a CO2/N2-mixture, thus mimicking initial CO2 concentrations in high-inoculum-density, nitrogen-sparged cultures, or by using l-aspartate instead of ammonium as nitrogen source. This study resolves apparent contradictions in the literature on the genetic interventions required for anaerobic growth of CEN.PK-derived strains on d-xylose. Additionally, it indicates the potential relevance of CO2 availability and anaplerotic carboxylation reactions for anaerobic growth of engineered S. cerevisiae strains on d-xylose., This study resolves apparent contradictions concerning the genetic interventions required for xylose fermentation by S. cerevisiae. Additionally, it indicates the potential relevance of CO2 and anaplerotic reactions for the latter.

Cell volume is an important parameter for modelling cellular processes. Temperature-induced variability of cellular size, volume, intracellular granularity, a fraction of budding cells of yeast Saccharomyces cerevisiae CEN.PK 113-7D (in anaerobic glucose unlimited batch cultures) were measured by flow cytometry and matched with the performance of the biomass growth (maximal specific growth rate (μmax), specific rate of glucose consumption, the rate of maintenance, biomass yield on glucose). The critical diameter of single cells was 7.94 μm and it is invariant at growth temperatures above 18.5°C. Below 18.5°C, it exponentially increases up to 10.2 μm. The size of the bud linearly depends on μmax, and it is between 50% at 5°C and 90% at 31°C of the averaged single cell. The intracellular granularity (side scatter channel (SSC)-index) negatively depends on μmax. There are two temperature regions (5-31°C vs. 33-40°C) where the relationship between SSC-index and various cellular parameters differ significantly. In supraoptimal temperature range (33-40°C), cells are less granulated perhaps due to a higher rate of the maintenance. There is temperature dependent passage through the checkpoints in the cell cycle which influences the μmax. The results point to the existence of two different morphological states of yeasts in these different temperature regions.

Cas9-assisted genome editing was used to construct an engineered glucose-phosphorylation-negative S. cerevisiae strain, expressing the Lactobacillus plantaruml-arabinose pathway and the Penicillium chrysogenum transporter PcAraT. This strain, which showed a growth rate of 0.26 h−1 on l-arabinose in aerobic batch cultures, was subsequently evolved for anaerobic growth on l-arabinose in the presence of d-glucose and d-xylose. In four strains isolated from two independent evolution experiments the galactose-transporter gene GAL2 had been duplicated, with all alleles encoding Gal2N376T or Gal2N376I substitutions. In one strain, a single GAL2 allele additionally encoded a Gal2T89I substitution, which was subsequently also detected in the independently evolved strain IMS0010. In 14C-sugar-transport assays, Gal2N376S, Gal2N376T and Gal2N376I substitutions showed a much lower glucose sensitivity of l-arabinose transport and a much higher Km for d-glucose transport than wild-type Gal2. Introduction of the Gal2N376I substitution in a non-evolved strain enabled growth on l-arabinose in the presence of d-glucose. Gal2N376T, T89I and Gal2T89I variants showed a lower Km for l-arabinose and a higher Km for d-glucose than wild-type Gal2, while reverting Gal2N376T, T89I to Gal2N376 in an evolved strain negatively affected anaerobic growth on l-arabinose. This study indicates that optimal conversion of mixed-sugar feedstocks may require complex ‘transporter landscapes’, consisting of sugar transporters with complementary kinetic and regulatory properties., This research describes the construction, laboratory evolution and characterization of l-arabinose fermenting Saccharomyces cerevisiae strains that are able to consume specifically l-arabinose in the presence of d-glucose and d-xylose.

One of the fundamental processes that determine cellular fate is regulation of gene transcription. Understanding these regulatory processes is therefore essential for understanding cellular responses to changes in environmental conditions. At the core promoter, the regulatory region containing the transcription start site (TSS), all inputs regulating transcription are integrated. Here, we used Cap Analysis of Gene Expression (CAGE) to analyze the pattern of transcription start sites at four different environmental conditions (limited in ethanol, limited in nitrogen, limited in glucose and limited in glucose under anaerobic conditions) using the Saccharomyces cerevisiae strain CEN.PK113-7D. With this experimental setup we were able to show that the TSS landscape in yeast is stable at different metabolic states of the cell. We also show that the spatial distribution of transcription initiation events, described by the shape index, has a surprisingly strong negative correlation with measured gene expression levels, meaning that genes with higher expression levels tend to have a broader distribution of TSSs. Our analysis supplies a set of high quality TSS annotations useful for metabolic engineering and synthetic biology approaches in the industrially relevant laboratory strain CEN.PK113-7D, and provides novel insights into yeast TSS dynamics and gene regulation.

D-Glucose, D-xylose and L-arabinose are major sugars in lignocellulosic hydrolysates. This study explores fermentation of glucose-xylose-arabinose mixtures by a consortium of three ‘specialist’ Saccharomyces cerevisiae strains. A D-glucose- and L-arabinose-tolerant xylose specialist was constructed by eliminating hexose phosphorylation in an engineered xylose-fermenting strain and subsequent laboratory evolution. A resulting strain anaerobically grew and fermented D-xylose in the presence of 20 g L-1 of D-glucose and L-arabinose. A synthetic consortium that additionally comprised a similarly obtained arabinose specialist and a pentose-non-fermenting laboratory strain, rapidly and simultaneously converted D-glucose and L-arabinose in anaerobic batch cultures on three-sugar mixtures. However, performance of the xylose specialist was strongly impaired in these mixed cultures. After prolonged cultivation of the consortium on three-sugar mixtures, the time required for complete sugar conversion approached that of a previously constructed and evolved ‘generalist’ strain. In contrast to the generalist strain, whose fermentation kinetics deteriorated during prolonged repeated-batch cultivation on a mixture of 20 g L-1 D-glucose, 10 g L-1 D-xylose and 5 g L-1 L-arabinose, the evolved consortium showed stable fermentation kinetics. Understanding the interactions between specialist strains is a key challenge in further exploring the applicability of this synthetic consortium approach for industrial fermentation of lignocellulosic hydrolysates.

Weak acids are present in lignocellulosic hydrolysate as potential inhibitors that can hamper the use of this renewable resource for fuel and chemical production. To study the effects of weak acids on yeast growth, physiological investigations were carried out in batch cultures using glucose as carbon source in the presence of acetic, formic, levulinic, and vanillic acid at three different concentrations at pH 5.0. The results showed that acids at moderate concentrations can stimulate the glycolytic flux, while higher levels of acid slow down the glycolytic flux for both aerobically and anaerobically grown yeast cells. In particular, the flux distribution between respiratory and fermentative growth was adjusted to achieve an optimal ATP generation to allow a maintained energy level as high as it is in nonstressed cells grown exponentially on glucose under aerobic conditions. In addition, yeast cells exposed to acids suffered from severe reactive oxygen species stress and depletion of reduced glutathione commensurate with exhaustion of the total glutathione pool. Furthermore, a higher cellular trehalose content was observed as compared to control cultivations, and this trehalose probably acts to enhance a number of stress tolerances of the yeast.

Squalene is a valuable natural substance with several biotechnological applications. In the yeast Saccharomyces cerevisiae, it is produced in the isoprenoid pathway as the first precursor dedicated to ergosterol biosynthesis. The aim of this study was to explore the potential of squalene epoxidase encoded by the ERG1 gene as the target for manipulating squalene levels in yeast. Highest squalene levels (over 1000 μg squalene per 10(9) cells) were induced by specific point mutations in ERG1 gene that reduced activity of squalene epoxidase and caused hypersensitivity to terbinafine. This accumulation of squalene in erg1 mutants did not significantly disturb their growth. Treatment with squalene epoxidase inhibitor terbinafine revealed a limit in squalene accumulation at 700 μg squalene per 10(9) cells which was associated with pronounced growth defects. Inhibition of squalene epoxidase activity by anaerobiosis or heme deficiency resulted in relatively low squalene levels. These levels were significantly increased by ergosterol depletion in anaerobic cells which indicated feedback inhibition of squalene production by ergosterol. Accumulation of squalene in erg1 mutants and terbinafine-treated cells were associated with increased cellular content and aggregation of lipid droplets. Our results prove that targeted genetic manipulation of the ERG1 gene is a promising tool for increasing squalene production in yeast.

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.

The Nakaseomyces clade consists of a group of six hemiascomyceteous yeasts (Candida glabrata, Nakaseomyces delphensis, Candida nivarensis, Candida bracarensis, Candida castelli, Nakaseomyces bacillisporus), phylogenetically close to the yeast Saccharomyces cerevisiae, their representative being the well-known pathogenic yeast Candida glabrata. Four species had been previously examined for their carbon assimilation properties and found to have similar properties to S. cerevisiae (repression of respiration in high glucose- i.e. Crabtree-positivity- and being a facultative anaerobe). We examined here the complete set of the six species for their carbon metabolic gene content. We also measured different metabolic and life-history traits (glucose consumption rate, population growth rate, carrying capacity, cell size, cell and biomass yield). We observed deviations from the glycolytic gene redundancy observed in S. cerevisiae presumed to be an important property for the Crabtree positivity, especially for the two species Candida castelli and Nakaseomyces bacillisporus which frequently have only one gene copy, but different life strategies. Therefore we show that the decrease in carbon metabolic gene copy cannot be simply associated to a reduction of glucose consumption rate and can be counterbalanced by other beneficial genetic variations.

Conversion of glucose to lactic acid is stoichiometrically equivalent to ethanol formation with respect to ATP formation from substrate-level phosphorylation, redox equivalents and product yield. However, anaerobic growth cannot be sustained in homolactate fermenting Saccharomyces cerevisiae. ATP-dependent export of the lactate anion and/or proton, resulting in net zero ATP formation, is suspected as the underlying cause. In an effort to understand the mechanisms behind the decreased lactic acid production rate in anaerobic homolactate cultures of S. cerevisiae, aerobic carbon-limited chemostats were performed and subjected to anaerobic perturbations in the presence of high glucose concentrations. Intracellular measurements of adenosine phosphates confirmed ATP depletion and decreased energy charge immediately upon anaerobicity. Unexpectedly, readily available sources of carbon and energy, trehalose and glycogen, were not activated in homolactate strains as they were in reference strains that produce ethanol. Finally, the anticipated increase in maximal velocity (V(max)) of glycolytic enzymes was not observed in homolactate fermentation suggesting the absence of protein synthesis that may be attributed to decreased energy availability. Essentially, anaerobic homolactate fermentation results in energy depletion, which, in turn, hinders protein synthesis, central carbon metabolism and subsequent energy generation.

The molecular mechanisms involved in the ability of cells to adapt and respond to differing oxygen tensions are of great interest to the pharmaceutical, medical and fermentation industries. The transcriptional profiles reported in previous studies of cells grown under anaerobic, aerobic and dynamic growth conditions have shown significantly altered responses including induction of genes regulated by the oxidative stress transcription factor Yap1p when oxygen was present. The present study investigated the phenotypic changes that occur in cells when shifted from anaerobic to aerobic growth conditions and it was found through mutant analyses that the elevated activity of Yap1p during the shift was mediated by the phospholipid hydroperoxide-sensing protein encoded by GPX3. Cell viability and growth rate were unaffected even though anaerobically grown cells were found to be hypersensitive to low doses of the oxidative stress-inducing compound hydrogen peroxide (H(2)O(2)). Adaptation to H(2)O(2) treatment was demonstrated to occur when anaerobically grown wild-type cells were aerated for a short time that was reliant on the Yap1p and Skn7p transcription factors.

The development of fermentative yeasts secreting no organic acids is highly desirable for ethanol production coupled with membrane separation processes, because the acidic byproduct, succinic acid, significantly inhibits the membrane permeation of ethanol. Of the Pichia and Candida yeasts tested, Candida krusei IA-1 showed the highest ethanol productivity [55 g L(-1) day(-1) from 150 g L(-1) (w/v) of glucose], comparable to the strains of Saccharomyces cerevisiae, and produced much less of the acid (0.6 g L(-1) day(-1)) than the Saccharomyces strains (1.5-1.8 g L(-1) day(-1)) under semi-aerobic conditions. Interestingly, under aerobic conditions, strain IA-1 showed no production of the acid. Stain IA-1 exhibited a good assimilation of the acid, while S. cerevisiae NBRC 0216 showed no assimilation. The activity of succinate dehydrogenase (SDH) in strain IA-1 was 37.5 mU mg(-1), and 7.8-fold higher than that in S. cerevisiae strain NBRC 0216. More significantly, SDH1 was abundantly transcribed in strain IA-1, different from that in strain NBRC 0216, regardless of the culture conditions. From these results, C. krusei IA-1 efficiently takes up succinic acid and metabolizes it in the Krebs cycle, producing an extremely low level of byproducts in the culture medium. Therefore, C. krusei is not only a promising alternative to S. cerevisiae but also a suitable model for metabolic engineering of S. cerevisiae.

Saccharomyces cerevisiae CEN.PK113-1A was grown in glucose-limited chemostat culture with 0%, 0.5%, 1.0%, 2.8% or 20.9% O2 in the inlet gas (D=0.10 h-1, pH 5, 30°C) to determine the effects of oxygen on 17 metabolites and 69 genes related to central carbon metabolism. The concentrations of tricarboxylic acid cycle (TCA) metabolites and all glycolytic metabolites except 2-phosphoglycerate+3-phosphoglycerate and phosphoenolpyruvate were higher in anaerobic than in fully aerobic conditions. Provision of only 0.5-1% O2 reduced the concentrations of most metabolites, as compared with anaerobic conditions. Transcription of most genes analyzed was reduced in 0%, 0.5% or 1.0% O2 relative to cells grown in 2.8% or 20.9% O 2. Ethanol production was observed with 2.8% or less O2. After steady-state analysis in defined oxygen concentrations, the conditions were switched from aerobic to anaerobic. Metabolite and transcript levels were monitored for up to 96 h after the transition, and this showed that more than 30 h was required for the cells to fully adapt to anaerobiosis. Levels of metabolites of upper glycolysis and the TCA cycle increased following the transition to anaerobic conditions, whereas those of metabolites of lower glycolysis generally decreased. Gene regulation was more complex, with some genes showing transient upregulation or downregulation during the adaptation to anaerobic conditions.

Dekkera bruxellensis is a non-conventional yeast normally considered a spoilage organism in wine (off-flavours) and in the bioethanol industry. But it also has potential as production yeast. The species diverged from Saccharomyces cerevisiae 200 mya, before the whole genome duplication. However, it displays similar characteristics such as being Crabtree- and petite positive, and the ability to grow anaerobically. Partial increases in ploidy and promoter rewiring may have enabled evolution of the fermentative lifestyle in D. bruxellensis. On the other hand, it has genes typical for respiratory yeasts, such as for complex I or the alternative oxidase AOX1. Dekkera bruxellensis grows more slowly than S. cerevisiae, but produces similar or greater amounts of ethanol, and very low amounts of glycerol. Glycerol production represents a loss of energy but also functions as a redox sink for NADH formed during synthesis of amino acids and other compounds. Accordingly, anaerobic growth required addition of certain amino acids. In spite of its slow growth, D. bruxellensis outcompeted S. cerevisiae in glucose-limited cultures, indicating a more efficient energy metabolism and/or higher affinity for glucose. This review tries to summarize the latest discoveries about evolution, physiology and metabolism, and biotechnological potential of D. bruxellensis.

A sealed fermentation (SF) system and an anaerobic fermentation (AF) system (under normal atmospheric pressure conditions) were employed to study the influence of endogenous carbon dioxide (CO 2) on the metabolism of Saccharomyces cerevisiae. The results showed that the fermentation stopped when 82.0 g L −1 glucose was consumed and the endogenously produced CO2: pressure reached to 14.3 MPa in SF system, while the sugar was used up during AF. The total yeast viable count in the end of AF was higher than that of SF. It was also observed that the ethanol yield in AF and SF was similar, the glycerol yield in AF was 1.26 times higher than that in SF, while the succinic acid and acetic acid yields in SF were 24.7 and 26 times higher than that in AF, respectively. Additionally, this work provides a stoichiometric model used for metabolic flux analysis of S. cerevisiae to compare the flux distribution in SF and AF. The results showed that CO 2 had an important effect on the pathways of oxaloacetic acid formation from pyruvic acid and ribose-5-phosphate formation from glucose-6-phosphate. However, the pathway of ethanol formation from pyruvic acid (decarboxylation reaction), catalyzed by pyruvate decarboxylase, was insensitive to CO2.

Anaerobic Saccharomyces cerevisiae cultures reoxidize the excess NADH formed in biosynthesis via glycerol production. This study investigates whether cometabolism of formate, a well-known NADH-generating substrate in aerobic cultures, can increase glycerol production in anaerobic S. cerevisiae cultures. In anaerobic, glucose-limited chemostat sultures (D=0.10 h(-1)) with molar formate-to-glucose ratios of 0 to 0.5, only a small fraction of the formate added to the cultures was consumed. To investigate whether incomplete formate consumption was by the unfavourable kinetics of yeast formate dehydrogenase (high k(M) for formate at low intracellular NAD(+) concentrations) strains were constructed in which the FDH1 and/or GPD2 genes, encoding formate dehydrogenase and glycerol-3-phosphate dehydrogenase, respectively, were overexpressed. The engineered strains consumed up to 70% of the formate added to the feed, thereby increasing glycerol yields to 0.3 mol mol(-1) glucose at a formate-to-glucose ratio of 0.34. In all strains tested, the molar ratio between formate consumption and additional glycerol production relative to a reference culture equalled one. While demonstrating that that format can be use to enhance glycerol yields in anaerobic S. cerevisiae cultures, This study also reveals kinetic constraints of yeast formate dehydrogenase as an NADH-generating system in yeast mediated reduction processes.

Although some yeast species, e.g. Saccharomyces cerevisiae, can grow under anaerobic conditions, Kluyveromyces lactis cannot. In a systematic study, we have determined which S. cerevisiae genes are required for growth without oxygen. This has been done by using the yeast deletion library. Both aerobically essential and nonessential genes have been tested for their necessity for anaerobic growth. Upon comparison of the K. lactis genome with the genes found to be anaerobically important in S. cerevisiae, which yielded 20 genes that are missing in K. lactis, we hypothesize that lack of import of sterols might be one of the more important reasons that K. lactis cannot grow in the absence of oxygen.

The KlAAC gene, encoding the ADP/ATP carrier, has been assumed to be a single gene in Kluyveromyces lactis, an aerobic, petite-negative yeast species. The Klaac null mutation, which causes a respiratory-deficient phenotype, was fully complemented by AAC2, the Saccharomyces cerevisiae major gene for the ADP/ATP carrier and also by AAC1, a gene that is poorly expressed in S. cerevisiae. In this study, we demonstrate that the Klaac null mutation is partially complemented by the ScAAC3 gene, encoding the hypoxic ADP/ATP carrier isoform, whose expression in S. cerevisiae is prevented by oxygen. Once introduced into K. lactis, the AAC3 gene was expressed both under aerobic and under partial anaerobic conditions but did not support the growth of K. lactis under strict anaerobic conditions.

Yeast species can grow on various sugars. However, in many cases the growth on certain sugars (especially oligosaccharides) occurs only under aerobic conditions, and not in anaerobiosis or in the absence of respiration. Fermentation is blocked under these conditions. This apparent dependence of sugar utilization on the respiration has been called Kluyver effect, and such 'respiration-dependent' species are called Kluyver effect positive. A yeast may be Kluyver effect positive for some sugars and not for others. The physiological meaning and the molecular basis of the phenomenon are not clear. It has recently been reported that Kluyveromyces lactis, which is Kluyver effect positive for galactose and a few other sugars, could be converted into a Kluyver effect-negative form by introduction of relevant sugar transporter genes. Such results offer for the first time a direct support to the hypothesis that the immediate cause of the Kluyver effect may be the low level of sugar transporter activities which is not sufficient to sustain the high substrate flow necessary for fermentative growth, whereas the energy-efficient respiratory growth does not require a high rate of sugar uptake. We examined to what extent this sugar transporter theory of the Kluyver effect can be generalized.

Under anaerobic conditions, Saccharomyces cerevisiae uses NADH-dependent glycerol-3-phosphate dehydrogenase (Gpd1p and Gpd2p) to re-oxidize excess NADH, yielding substantial amounts of glycerol. In a Deltagpd1 Deltagpd2 double-null mutant, the necessary NAD+ regeneration through glycerol production is no longer possible, and this mutant does not grow under anaerobic conditions. The excess NADH formed can potentially be used to drive other NADH-dependent reactions or pathways. To investigate this possibility, a double-null mutant was transformed with a heterologous gene (mtlD) from Escherichia coli, coding for NADH-dependent mannitol-1-phosphate dehydrogenase. Expression of this gene in S. cerevisiae should result in NADH oxidation by the NADH-requiring formation of mannitol-1-phosphate from fructose-6-phosphate. The strain was characterized using step-change experiments, in which, during the exponential growth phase, the inlet gas was changed from air to nitrogen. It was found that the mutant produced mannitol only under anaerobic conditions. However, anaerobic growth was not regained, which was probably due to the excessive accumulation of mannitol in the cells.

Mucor circinelloides (syn. racemosus) is a non-pathogenic dimorphic fungus belonging to the class of zygomycetes. We are developing a novel system for heterologous protein production exploiting the dimorphic growth characteristics of M. circinelloides. In order to identify potential genetic regulators of morphology we have initiated a characterisation of key genes involved in signal transduction in Mucor. We have cloned and characterised pkaR and pkaC encoding the regulatory subunit (PKAR) and the catalytic subunit (PKAC), respectively, of the cAMP-dependent protein kinase A (PKA) of M. circinelloides. In anaerobically grown yeast cells, the levels of expression of both pkaR and pkaC were significantly higher than the levels of expression in aerobically grown mycelium. However, during the dimorphic shift, i.e. during the transition from anaerobic yeast growth to aerobic filamentous growth, the expression of pkaR was found to increase approximately two-fold. These results indicate that regulation of PKA activity is conferred at different levels according to growth and environmental conditions. Overexpression of pkaR resulted in a multi-branched colony phenotype on solid medium indicating that PKAR plays a role in filamentation and branching. Fragments of genes encoding factors of the mitogen-activated protein (MAP) kinase (MAPK) pathway have also been cloned: mpk1 (mitogen-activated protein kinase 1) encoding a MAPK homologue and ste12 encoding a transcription factor.

Sterol uptake in fungi is a multistep process that involves interaction between external sterols and the cell wall, incorporation of sterol molecules into the plasma membrane, and subsequent integration into intracellular membranes for turnover. ATP-binding cassette (ABC) transporters have been implicated in sterol uptake, but key features of their activity remain to be elucidated. Here, we apply fluorescent cholesterol (NBD-cholesterol) to monitor sterol uptake under anaerobic and aerobic conditions in two fungal species, Candida glabrata (Cg) and Saccharomyces cerevisiae (Sc). We found that in both fungal species, ABC transporter-dependent uptake of cholesterol under anaerobic conditions and in mutants lacking HEM1 gene is promoted in the presence of the serum protein albumin that is able to bind the sterol molecule. Furthermore, the C. glabrata ABC transporter CgAus1p expressed in S. cerevisiae requires the presence of serum or albumin for efficient cholesterol uptake. These results suggest that albumin can serve as sterol donor in ABC transporter-dependent sterol uptake, a process potentially important for growth of C. glabrata inside infected humans.

Selected Saccharomyces cerevisiae strains are used in Brazil to produce the hitherto most energetically efficient first-generation fuel ethanol. Although genome and some transcriptome data are available for some of these strains, quantitative physiological data are lacking. This study investigates the physiology of S. cerevisiae strain PE-2, widely used in the Brazilian fuel ethanol industry, in comparison with CEN.PK113-7D, a reference laboratory strain, focusing on tolerance to low pH and acetic acid stress. Both strains were grown in anaerobic bioreactors, operated as batch, chemostat or dynamic continuous cultures. Despite their different backgrounds, biomass and product formation by the two strains were similar under a range of conditions (pH 5 or pH < 3, with or without 105 mM acetic acid added). PE-2 displayed a remarkably higher fitness than CEN.PK113-7D during batch cultivation on complex Yeast extract - Peptone - Dextrose medium at low pH (2.7). Kinetics of viability loss of non-growing cells, incubated at pH 1.5, indicated a superior survival of glucose-depleted PE-2 cells, when compared with either CEN.PK113-7D or a commercial bakers' strain. These results indicate that the sulfuric acid washing step, used in the fuel ethanol industry to decrease bacterial contamination due to non-aseptic operation, might have exerted an important selective pressure on the microbial populations present in such environments.

During alcoholic fermentation, Saccharomyces cerevisiae is exposed to continuously changing environmental conditions, such as decreasing sugar and increasing ethanol concentrations. Oxygen, a critical nutrient to avoid stuck and sluggish fermentations, is only discretely available throughout the process after pump-over operation. In this work, we studied the physiological response of the wine yeast S. cerevisiae strain EC1118 to a sudden increase in dissolved oxygen, simulating pump-over operation. With this aim, an impulse of dissolved oxygen was added to carbon-sufficient, nitrogen-limited anaerobic continuous cultures. Results showed that genes related to mitochondrial respiration, ergosterol biosynthesis, and oxidative stress, among other metabolic pathways, were induced after the oxygen impulse. On the other hand, mannoprotein coding genes were repressed. The changes in the expression of these genes are coordinated responses that share common elements at the level of transcriptional regulation. Beneficial and detrimental effects of these physiological processes on wine quality highlight the dual role of oxygen in ‘making or breaking wines’. These findings will facilitate the development of oxygen addition strategies to optimize yeast performance in industrial fermentations.

The removal of Gal80 protein by gene disruption turned into efficient GAL promoter-driven heterologous gene expression under anaerobic alcoholic fermentation of Saccharomyces cerevisiae. Using lipase B from Candida antarctica as a reporter, the relative strength of GAL10 promoter (P(GAL10) ) in Δgal80 mutant that does not require galactose as an inducer was compared to those of ADH1, PDC1, and PGK promoters, which have been known to work well anaerobically in actively fermenting yeast cells under high glucose concentration. P(GAL10) in the Δgal80 mutant showed 0.8-fold (ADH1), fourfold (PDC1), and 50-fold (PGK) in promoter strength.

Yeast species belonging to the lineage that underwent the whole genome duplication (WGD), and including Saccharomyces cerevisiae, can grow under anaerobiosis and accumulate ethanol in the presence of glucose and oxygen. The pre-WGD yeasts, which branched from the S. cerevisiae lineage just before the WGD event, including Kluyveromyces lactis, are more dependent on oxygen and do not accumulate large amounts of ethanol in the presence of excess oxygen. Yeasts that belong to the so-called 'lower branches' of the yeast phylogenetic tree and diverged from S. cerevisiae more than 200 million years ago have so far not been thoroughly investigated for their physiology and carbon metabolism. Here, we have studied several isolates of Candida albicans and Debaryomyces hansenii for their dependence on oxygen. Candida albicans grew very poorly at an oxygen concentration1 p.p.m. and D. hansenii could not grow at all. In aerobic batch cultivations, C. albicans exhibited a predominantly aerobic metabolism, accumulating only small amounts of ethanol (0.01-0.09 g g(-1) glucose). Apparently, C. albicans and several other pre-WGD yeasts still exhibit the original traits of the yeast progenitor: poor accumulation of ethanol under aerobic conditions and strong dependence on the presence of oxygen.

Transcriptome and proteome profiles have been established for the commercial wine yeast strain T73 during an important industrial process: yeast biomass propagation. The data from both analyses reveal that the metabolic transition from fermentation to respiration is the most critical step in biomass propagation. We identified 177 ORFs and 56 proteins among those most expressed during the process, thus highlighting cell stress response, mitochondrial and carbohydrate metabolism as the most represented functional categories. A direct correlation between mRNA changes and protein abundance was observed for several functional categories such as tricarboxylic acid cycle proteins, heat shock proteins, chaperons and oxidative stress response-related proteins. However, we found no concordance in the transcript and proteomic levels for glycolytic proteins, which is probably due to post-translational modifications increasing the number of protein isoforms, especially at the end of biomass propagation. The correlation between protein abundance and the enzyme activities of alcohol dehydrogenase, pyruvate decarboxylase and glyceraldehyde-3-phosphate dehydrogenase was not affected by these modifications. We suggest post-translational mechanisms during biomass propagation that affect the stability of those proteins that play an important role in the produced biomass' fermentative capacity.

The constructed strains AM63, having an extra copy of the HMG2 gene with a K6R stabilizing mutation in Hmg2p expressed under the control of the inducible galactose promoter and stably integrated into the chromosomal HO locus, and AM64, a derivative of AM63 with an additional deletion of the ERG6 gene, were used as tools to test the squalene accumulation capacity of Saccharomyces cerevisiae. Kinetic data indicated high squalene levels in the early stages of semi-anaerobic cultivation of these strains. The stable Hmg2p induced a strong increase in the squalene pool and a smaller increase in the lanosterol pool. In AM63, the squalene content was approximately 20-fold higher than in the wild-type EGY48 parental strain. In AM64, the combined Hmg2p stabilization and ERG6 deletion did not further enhance squalene accumulation, as lack of ergosterol feedback inhibition led to an elevated transfer of surplus squalene into C27 sterols. The obtained maximum capacity of the selected strains to accumulate squalene and our observations provide a further understanding of the regulation of ergosterol pathway and may also be used as a reference value for its production using food-grade strains of S. cerevisiae.