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The hexose transporters of Saccharomyces cerevisiae play different roles during enological fermentation
Abstract
We investigated the role of hexose transporters in a Saccharomyces cerevisiae strain derived from an industrial wine strain by carrying out a functional analysis of HXT genes 1-7 under enological conditions. A strain in which the sugar carrier genes HXT1-HXT7 were deleted was constructed and the HXT genes were expressed individually or in combination to evaluate their role under wine alcoholic fermentation conditions. No growth or fermentation was observed in winemaking conditions for the hxt1-7 delta strain. The low-affinity carriers Hxt1 and Hxt3 were the only carriers giving complete fermentation of sugars when expressed alone, indicating that these carriers play a predominant role in wine fermentation. However, these two carriers have different functions. The Hxt3 transporter is thought to play a major role, as it was the only carrier that gave an almost normal fermentation profile when produced alone. The hxt1 carrier was much less effective during the stationary phase and its role is thought to be restricted to the beginning of fermentation. The high-affinity carriers Hxt2, Hxt6 and/or Hxt7 were also required for normal fermentation. These high-affinity transporters have different functions: hxt2 is involved in growth initiation, whereas Hxt6 and/or Hxt7 are required at the end of alcoholic fermentation. This work shows that the successful alcoholic fermentation of wine involves at least four or five hexose carriers, playing different roles at various stages in the fermentation cycle.
... In fact, Hxt3p is the only carrier that is expressed throughout the fermentation, consistent with the fact that it plays a key role in the process [26]. The robust expression and high stability of Hxt3p during the stationary phase is consistent with its capacity to maintain a high fermentation rate during starvation when expressed alone [7]. ...
... The transport of glucose and fructose in the yeast Saccharomyces cerevisiae plays a crucial role in controlling the rate of wine fermentation, and the yeast fermentation performance is strongly influenced by the hexose carrier set-up [7,26,42]. Saccharomyces cerevisiae is the organism provided with the highest number of hexose transporter genes. In its genome, twenty genes encode hexose transporter-like proteins (HXT1 to HXT17, GAL2, SNF3, and RGT2) [43]. ...
... This led to rethinking the importance of the HXT3 that was previously consid-ered to play a major role in sugar transport during the overall fermentation process. The main contribution of HXT3 to maintain a high fermentation ratio during late fermentation phase [7,40] could be limited to specific yeast genotypes. In different genetic contexts, other genes such as HXT6/7, HXT8, and FSY1 were the most expressed and therefore responsible for sugar transport in the late fermentation phase. ...
In Saccharomyces cerevisiae, the fermentation rate and the ability to complete the sugar transformation process depend on the glucose and fructose transporter set-up. Hexose transport mainly occurs via facilitated diffusion carriers and these are encoded by the HXT gene family and GAL2. In addition, FSY1, coding a fructose/H+ symporter, was identified in some wine strains. This little-known transporter could be relevant in the last part of the fermentation process when fructose is the most abundant sugar. In this work, we investigated the gene expression of the hexose transporters during late fermentation phase, by means of qPCR. Four S. cerevisiae strains (P301.9, R31.3, R008, isolated from vineyard, and the commercial EC1118) were considered and the transporter gene expression levels were determined to evaluate how the strain gene expression pattern modulated the late fermentation process. The very low global gene expression and the poor fermentation performance of R008 suggested that the overall expression level is a determinant to obtain the total sugar consumption. Each strain showed a specific gene expression profile that was strongly variable. This led to rethinking the importance of the HXT3 gene that was previously considered to play a major role in sugar transport. In vineyard strains, other transporter genes, such as HXT6/7, HXT8, and FSY1, showed higher expression levels, and the resulting gene expression patterns properly supported the late fermentation process.
... /an notation /genome/ saccharomyces_cerevisiae.3/Home.html) and to published data for strain V5 (Luyten et al. 2002) (Table 1 ). Four isolates from independent wineries were used: UCD932, UCD935, UCD939, and UCD940 (Mor timer et al. 1994 ). ...
... There were no other amino acid substitutions found in Hxt4p for these strains. However, the V5 strain was shown to contain a nonsense mutation in HXT4 and does not express this protein (Luyten et al. 2002). Similarly, the RM11-1a strain displays multiple in-frame stop codons in HXT4 (Figure 2), and it would not be expected to express a functional protein. ...
... Perez et al. (2005) also noted expression of Hxt2p and suggested that the transient expression of Hxt2p during lag phase of the fermentation indicated a role for the protein in growth initiation. The authors did not observe Hxt4p since the HXT4 gene of yeast strain V5 has been shown to contain a nonsense mutation and does not express a functional carrier (Luyten et al. 2002). However, expression of Hxt4p also was not observed in our study. ...
Saccharomyces cerevisiae maintains a large family of hexose transporters encoded by the HXT genes. The major transporter genes, HXT1 through HXT7, were sequenced from four vineyard isolates and two com- mercial wine yeast strains and compared to the sequences in the Saccharomyces Genome Database for strain S288C and to those available for two additional wine strains V5 and RM11-1a. Base pair changes leading to dif- ferences in amino acid sequence were found for all seven transporters. Differences ranged from none to eight amino acid variations for the sequenced strains, depending upon the strain and the gene, in comparison with S288C. In contrast, RM11-1a displayed high degeneracy with multiple in-frame stop mutations for HXT1, HXT4, and HXT6. Several wine strain sequences for the HXT4 gene contained an identical additional 16 amino acids at the C-terminus. Transporter protein levels were analyzed in a wine yeast strain (UCD932) using green f luo- rescent protein tagging. HXT5, not shown to be expressed in previous studies, was expressed in UCD932 during fermentation. Expression of HXT4, a prominently expressed transporter in laboratory media, was not detected. Deletion of HXT1, HXT3, or HXT5 did not result in a discernable phenotype in UCD932 under the fermentation conditions used in this study as compared with the wild type strain. However, the strain lacking HXT3 was un- able to complete the fermentation in media containing 5% exogenous ethanol. This result suggests that correct expression of HXT3 may play a role in ethanol tolerance.
... Expression of the genes encoding these transporters is regulated not only by the available sugar concentration [17,20,21] but also by osmotic pressure [22,23], starvation [24] and the physiological state of the cell [21,25,26,27]. This complex regulation ensures that the yeast receives an adequate supply of carbon and energy under various conditions [28]. ...
... Experiments were conducted in order to determine if glucose and ethanol regulate the transcription of the F. oxysporum Hxt gene, as they do for yeast Hxt genes [17,28]. The Hxt mRNA levels were higher when the fungus was grown in 10 as compared to either 30 or 100 mM glucose as the sole carbon source ( Figure 3A). ...
... By changing the membrane lipid environment, ethanol indirectly inhibits the activity of various transport systems [39,40]. The rate of hexose transportation is determined by both the activity and the number of hexose transporters in the plasma membrane [28], hence with increasing ethanol concentration F. oxysporum cells try to compensate for the reduction in Hxt activity by increasing their numbers. During the bioconversion of straw to ethanol by F. oxysporum the amount of glucose in the broth remains very low (unpubl. ...
Ethanol is the most-widely used biofuel in the world today. Lignocellulosic plant biomass derived from agricultural residue can be converted to ethanol via microbial bioprocessing. Fungi such as Fusarium oxysporum can simultaneously saccharify straw to sugars and ferment sugars to ethanol. But there are many bottlenecks that need to be overcome to increase the efficacy of microbial production of ethanol from straw, not least enhancement of the rate of fermentation of both hexose and pentose sugars. This research tested the hypothesis that the rate of sugar uptake by F. oxysporum would enhance the ethanol yields from lignocellulosic straw and that high affinity glucose transporters can enhance ethanol yields from this substrate. We characterized a novel hexose transporter (Hxt) from this fungus. The F. oxysporum Hxt represents a novel transporter with homology to yeast glucose signaling/transporter proteins Rgt2 and Snf3, but it lacks their C-terminal domain which is necessary for glucose signalling. Its expression level decreased with increasing glucose concentration in the medium and in a glucose uptake study the Km((glucose)) was 0.9 mM, which indicated that the protein is a high affinity glucose transporter. Post-translational gene silencing or over expression of the Hxt in F. oxysporum directly affected the glucose and xylose transport capacity and ethanol yielded by F. oxysporum from straw, glucose and xylose. Thus we conclude that this Hxt has the capacity to transport both C5 and C6 sugars and to enhance ethanol yields from lignocellulosic material. This study has confirmed that high affinity glucose transporters are ideal candidates for improving ethanol yields from lignocellulose because their activity and level of expression is high in low glucose concentrations, which is very common during the process of consolidated processing.
... The fructophilic phenotype of some S. cerevisiae isolates has been associated with the presence of specific functional hexose transporters, as these are involved in the first steps of simple sugar catabolism by introducing the available hexoses throughout the cellular membrane (Reifenberger et al. 1997). In S. cerevisiae, 18 different hexose transporter proteins have been reported, in addition to two closely related glucose sensors, Rgt2p and Snf3p (Luyten et al. 2002), which share similar terminal amino acid sequences. Hexose transporter proteins 1-4, 6 and 7 are the main carriers reported in S. cerevisiae and can take up glucose, fructose and mannose (Reifenberger et al. 1997), thereby displaying diverse hexose specificities and affinities (Guillaume et al. 2007;Saloheimo et al. 2007). ...
... Hexose transporter proteins 1-4, 6 and 7 are the main carriers reported in S. cerevisiae and can take up glucose, fructose and mannose (Reifenberger et al. 1997), thereby displaying diverse hexose specificities and affinities (Guillaume et al. 2007;Saloheimo et al. 2007). Of all functional carriers reported, Hxt1p and Hxt3p seem to be the most relevant from the enological point of view, due to the high hexose (glucose and fructose) concentrations typical of wine musts (Luyten et al. 2002). Both Hxt1p and Hxt3p have been shown to have a low affinity for glucose, with K m values of approximately 100 and 60 mM, respectively (Reifenberger et al. 1997), and the peaks of gene expression profiles coincide with a higher rate of fructose consumption (Guillaume et al. 2007;Karpel et al. 2008). ...
... Both Hxt1p and Hxt3p have been shown to have a low affinity for glucose, with K m values of approximately 100 and 60 mM, respectively (Reifenberger et al. 1997), and the peaks of gene expression profiles coincide with a higher rate of fructose consumption (Guillaume et al. 2007;Karpel et al. 2008). Moreover, the expression of these two transporters alone is enough to achieve a full synthetic wine fermentation, demonstrated in the enologically-derived strain V5 (Luyten et al. 2002). This finding has been related to the fact that the HXT1 gene expression peaks during the lag phase and early exponential phase (high hexose concentrations) during wine fermentation (Luyten et al. 2002;Karpel et al. 2008), while HXT3 is expressed at the onset of the stationary phase (Luyten et al. 2002;Maier et al. 2002) when the ethanol concentration is already high and deleterious to cell membranes and proteins, including most carriers. ...
Mezcal from Tamaulipas (México) is produced by spontaneous alcoholic fermentation using Agave spp. musts, which are rich in fructose. In this study eight Saccharomyces cerevisiae isolates obtained at the final stage of fermentation from a traditional mezcal winery were analysed in three semi-synthetic media. Medium M1 had a sugar content of 100 g l(-1) and a glucose/fructose (G/F) of 9:1. Medium M2 had a sugar content of 100 g l(-1) and a G/F of 1:9. Medium M3 had a sugar content of 200 g l(-1) and a G/F of 1:1. In the three types of media tested, the highest ethanol yield was obtained from the glucophilic strain LCBG-3Y5, while strain LCBG-3Y8 was highly resistant to ethanol and the most fructophilic of the mezcal strains. Strain LCBG-3Y5 produced more glycerol (4.4 g l(-1)) and acetic acid (1 g l(-1)) in M2 than in M1 (1.7 and 0.5 g l(-1), respectively), and the ethanol yields were higher for all strains in M1 except for LCBG-3Y5, -3Y8 and the Fermichamp strain. In medium M3, only the Fermichamp strain was able to fully consume the 100 g of fructose l(-1) but left a residual 32 g of glucose l(-1). Regarding the hexose transporters, a high number of amino acid polymorphisms were found in the Hxt1p sequences. Strain LCBG-3Y8 exhibited eight unique amino acid changes, followed by the Fermichamp strain with three changes. In Hxt3p, we observed nine amino acid polymorphisms unique for the Fermichamp strain and five unique changes for the mezcal strains.
... Since the rate of anaerobic fermentation by yeast is limited primarily by the rate of sugar uptake (especially during the final fermentation stages of nitrogen depletion coupled to nutrient limitation), several laboratories focus on phosphorylation by the HXK1and HXK2-encoded hexokinases and the GLK1-encoded glucokinase, as well as on hexose sensors (encoded by SNF3 and RGT2) and hexose transporters (encoded by HXT1-HXT18 and GAL2) of S. cerevisiae (Kruckeberg 1996;Boles and Hollenberg 1997). At least five of these hexose transporters (Hxt1-3, Hxt6-7) of S. cerevisiae are required for a successful wine fermentation, playing different roles at various stages in the fermentation cycle (Luyten et al. 2002). The low-affinity transporters (Hxt1 and Hxt3) play a predominant role in wine fermentation; Hxt3 plays a major role, while Hxt1's role is restricted to the beginning of the wine fermentation (Luyten et al. 2002). ...
... At least five of these hexose transporters (Hxt1-3, Hxt6-7) of S. cerevisiae are required for a successful wine fermentation, playing different roles at various stages in the fermentation cycle (Luyten et al. 2002). The low-affinity transporters (Hxt1 and Hxt3) play a predominant role in wine fermentation; Hxt3 plays a major role, while Hxt1's role is restricted to the beginning of the wine fermentation (Luyten et al. 2002). The high-affinity carriers (Hxt2, Hxt6 and Hxt7) are also required at different stages of wine fermentation; Hxt2 is involved in growth initiation, whereas Hxt6 and Hxt7 are required at the end of alcoholic fermentation (Luyten et al. 2002). ...
... The low-affinity transporters (Hxt1 and Hxt3) play a predominant role in wine fermentation; Hxt3 plays a major role, while Hxt1's role is restricted to the beginning of the wine fermentation (Luyten et al. 2002). The high-affinity carriers (Hxt2, Hxt6 and Hxt7) are also required at different stages of wine fermentation; Hxt2 is involved in growth initiation, whereas Hxt6 and Hxt7 are required at the end of alcoholic fermentation (Luyten et al. 2002). ...
An urgent need has arisen to develop starter culture strains of Saccharomyces cerevisiae possessing a wide range of specialised properties in order to meet the new and challenging demands of the various wine producers
and consumers. Strain development is no longer limited to the primary role of wine yeasts, namely to catalyse the rapid and
complete conversion of grape sugars to alcohol and carbon dioxide without distorting the flavour of the final product. Today,
there is a much stronger emphasis on the development of wine yeasts for the cost-effective production of wine with minimised
resource inputs, improved quality and low environmental impact. This chapter focuses on the genetic constitution, analysis,
and improvement of wine yeasts and the potential role that customised starter yeast strains could play in improving the fermentation,
processing and biopreservation of wines, their capacity to enhance the wholesomeness and sensory quality of wine, and their
current status and future.
... Hexose transport in S. cerevisiae occurs only via facilitated diffusion carriers and these are encoded by several genes, including the HXT genes (Kruckeberg, 1996;Lagunas, 1993). There are 17 HXT genes in S. cerevisiae, but only seven of these, Hxt1p-Hxt7p, are required for growth on glucose or fructose (Boles & Hollenberg, 1997;Luyten et al., 2002;Reifenberger et al., 1997). The proteins encoded by these seven genes have different kinetic properties (K m from 1 to 300 mM) and are subject to different modes of regulation. ...
... To examine the functionality of the FSY1 gene, we expressed FSY1 under the control of the HXT3 promoter, in strain V5 hxt1-7D. This strain lacks the hexose transporter genes HXT1 to HXT7 and cannot utilize glucose, fructose or sucrose as sole carbon source (Luyten et al., 2002). As S. pastorianus Fsy1p has a low K m for fructose (Gonçalves et al., 2000), transformants were selected on minimal medium containing uracil and either a low concentration of fructose (0.5 or 1 %) or 2 % sucrose (which was converted into small amounts of glucose and fructose by invertase). ...
... The discrepancy in glucose and fructose utilization by S. cerevisiae is an essential step during oenological fermentation. The role and importance of sugar transport in oenological conditions were emphasized by Luyten et al. (2002), who showed that HXT1, HXT2, HXT3, HXT6 and HXT7 are required for optimal sugar fermentation in grape must. On the other hand, Guillaume et al. (2007) showed that the higher fructose utilization capacity of the Fermichamp wine yeast strain results from the expression of an allelic variant of HXT3. ...
Transport of glucose and fructose in the yeast Saccharomyces cerevisiae plays a crucial role in controlling the rate of wine fermentation. In S. cerevisiae, hexoses are transported by facilitated diffusion via hexose carriers (Hxt), which prefer glucose to fructose. However, utilization of fructose by wine yeast is critically important at the end of fermentation. Here, we report the characterization of a fructose transporter recently identified by sequencing the genome of the commercial wine yeast strain EC1118 and found in many other wine yeasts. This transporter is designated Fsy1p because of its homology with the Saccharomyces pastorianus fructose/H(+) symporter Fsy1p. A strain obtained by transformation of the V5 hxt1-7Δ mutant with FSY1 grew well on fructose, but to a much lesser extent on glucose as the sole carbon source. Sugar uptake and symport experiments showed that FSY1 encodes a proton-coupled symporter with high affinity for fructose (K(m) 0.24±0.04mM). Using real-time RT-PCR, we also investigated the expression pattern of FSY1 in EC1118 growing on various carbon sources. FSY1 was repressed by high concentrations of glucose or fructose and was highly expressed on ethanol as the sole carbon source. The characteristics of this transporter indicate that its acquisition could confer a significant advantage to S. cerevisiae during the wine fermentation process. This transporter is a good example of acquisition of a new function in yeast by horizontal gene transfer.
... The expression of these specific transporters depends on the concentration of glucose in the medium. High affinity transporters like Hxt6 and Hxt7 are repressed by high concentration of glucose, whereas transporters with low affinity such as Hxt1 and Hxt3 are induced by presence of high concentration of glucose (Rolland et al., 2002;Luyten et al., 2002). The transporters with intermediate affinity for glucose like Hxt2 and Hxt4 are iduced by low concentration of glucose and repressed by high concentration of glucose (Rolland et al., 2002). ...
... The transporters with intermediate affinity for glucose like Hxt2 and Hxt4 are iduced by low concentration of glucose and repressed by high concentration of glucose (Rolland et al., 2002). Luyten et al., (2002) had carried out functional analysis of the HXT1-7 genes to investigate the role of hexose transporter proteins in synthetic must. The deletion of HXT2 gene resulted only in a delayed start of fermentation and did not affect fermentation profile. ...
... The deletion of HXT2 gene resulted only in a delayed start of fermentation and did not affect fermentation profile. This suggested that Hxt2 transporter protein is involved in lag phase initiation of growth despite high concentration of sugar in the medium, which might be expected to repress expression of the HXT2 gene (Luyten et al., 2002). Mutant carried HXT3 gene only with other genes deleted were able to grow on glucose containing media regardless of the concentration of glucose (Özcan and Johnston, 1995;Luyten et al., 2002). ...
In the wine industry, the importance of selecting an appropriate yeast strain, generally of the species Saccharomyces cerevisiae, to ensure reliable fermentation and to achieve a desired level of quality has been well established. As a consequence, the demand for new starter cultures with improved or new oenological characteristics is increasing. Appropriately selected starter cultures can reduce the occurrence of stuck fermentations, impart specific aroma profiles and reduce the development of offflavours. Using standard breeding and selection procedures, several wine yeast strains that would be less likely than currently existing strains to experience stuck fermentation have previously been developed at the Institute for Wine Biotechnology. The target of these projects had been to develop strains with improved nitrogen efficiency [defined as the amount of fermented hexoses for a given amount of free amino nitrogen (FAN)], improved fructose utilization and ethanol tolerance. These three parameters are known contributors to stuck fermentation. Two of the strains that had been isolated in these projects, strain 116 for nitrogen efficiency and strain 38-1 for efficient fructose utilization, were chosen as parental strains for the current study. The aim was to further improve and possibly combine these traits in yeast strains by using hybridization followed by various enrichment and directed evolution procedures in a continuous fermentation setup. The strategy was to sequentially subject the population of mass-mated hybrids to a number of selective environments for a large number of generations. The yeasts were subjected to a high fructose/glucose ratio for 12 generations, followed by selection in an environment with a limited supply of nitrogen for 54 generations and finally to high ethanol stress. After each round of enrichment, individual strains were analysed to assess the results. For the hybrid strains selected after enrichment in a medium with a high fructose/glucose ratio, no general improvement could be discerned. However, one of the hybrids, hybrid strain 331, fermented fructose better than the parental strains and other hybrid strains. These results may suggest that the selection pressure was not applied for a sufficient number of generations and may not have been sufficiently strong. In addition, the parental strain may already performing at a rate that may render further improvement more difficult in this genetic background. The next aim of this study was to enhance fermentation performance of wine yeast hybrid strains in low nitrogen and high sugar conditions. Several hybrid strains 331, RR03 and 05R generated in this study showed improvement in efficiency of nitrogen utilization when compared to the parental strains, indicating a successful selection strategy. Several strains also showed higher ethanol tolerance, and some strains possessed] combinations of the traits to be improved. Future research will evaluate these hybrids regarding the production of aromatic compounds and of the sensory profile produced. Such strains would help the wine industry to control the occurrence of stuck fermentations and to produce quality wines. Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2008.
... Considering only glucose and fructose, transport has been pinpointed as a critical step for the different behaviors observed in the metabolism of such hexoses in S. cerevisiae (Luyten, Riou and Blondin 2002;Berthels et al. 2004). Among all the 20 proteins that constitute the group of hexose transporters and glucose sensors present in this microbe (Kruckeberg 1996;Özcan and Johnston 1999), Hxt1p to Hxt7p are the main ones in the context of glucose and/or fructose catabolism (Kruckeberg 1996;Luyten, Riou and Blondin 2002;Verwaal et al. 2002;Guillaume et al. 2007;Karpel, Place and Bisson 2008). ...
... Considering only glucose and fructose, transport has been pinpointed as a critical step for the different behaviors observed in the metabolism of such hexoses in S. cerevisiae (Luyten, Riou and Blondin 2002;Berthels et al. 2004). Among all the 20 proteins that constitute the group of hexose transporters and glucose sensors present in this microbe (Kruckeberg 1996;Özcan and Johnston 1999), Hxt1p to Hxt7p are the main ones in the context of glucose and/or fructose catabolism (Kruckeberg 1996;Luyten, Riou and Blondin 2002;Verwaal et al. 2002;Guillaume et al. 2007;Karpel, Place and Bisson 2008). Expression of these proteins is strain dependent (Verwaal et al. 2002;PEREZ et al. 2005) and their substrate affinities vary from one transporter to the other (Ozcan and Johnston 1995). ...
Present knowledge on the quantitative aerobic physiology of the yeast Saccharomyces cerevisiae during growth on sucrose as sole carbon and energy source is limited to either adapted cells or to the model laboratory strain CEN.PK113-7D. To broaden our understanding of this matter and open novel opportunities for sucrose-based biotechnological processes, we characterized three strains, with distinct backgrounds, during aerobic batch bioreactor cultivations. Our results reveal that sucrose metabolism in S. cerevisiae is a strain-specific trait. Each strain displayed a distinct extracellular hexose concentration and invertase activity profiles. Especially, the inferior maximum specific growth rate (0.21 h−1) of the CEN.PK113-7D strain, with respect to that of strains UFMG-CM-Y259 (0.37 h−1) and JP1 (0.32 h−1), could be associated to its low invertase activity (0.04 to 0.09 U mgDM−1). Moreover, comparative experiments with glucose or fructose alone, or in combination, suggest mixed mechanisms of sucrose utilization by the industrial strain JP1, and points out the remarkable ability of the wild isolate UFMG-CM-259 to grow faster on sucrose than on glucose in a well-controlled cultivation system. This work hints to a series of metabolic traits that can be exploited to increase sucrose catabolic rates and bioprocess efficiency.
... This finding was intriguing because some wine strains were previously found to have an impairment in high affinity hexose transport (the type of transporters expressed under the low sugar concentrations measured in olive brine), a trait that was subsequently associated to certain variants of the HXT hexose transporter genes (Luyten et al., 2002). Numerous HXT genes are present in the genomes of the species of the genus Saccharomyces, encoding transporters with different affinities for their substrates. ...
... Gene ontology analysis of the set of S. paradoxus genes present in homozygosity in all hybrid strains examined here, suggested that cell wall function and hexose transport were likely among the cellular processes benefiting from the S. paradoxus genomic contribution. Because inefficient high affinity hexose transport was found to be associated with specific HXT alleles carried by some wine strains (Luyten et al., 2002), we investigated whether the S. paradoxus HXT alleles retained by the olives strains were likely to contribute to the observed superior ability of these strains to consume sugars in brine. However, our experiments indicate that sugar consumption profiles are different between members of the Olives and Wine populations solely in the presence of NaCl and only when the cells are cultivated in nutritional conditions that support adaptation of their proteome to the high salt environment. ...
The adaptation of the yeast Saccharomyces cerevisiae to man-made environments for the fermentation of foodstuffs and beverages illustrates the scientific, social, and economic relevance of microbe domestication. Here we address a yet unexplored aspect of S. cerevisiae domestication, that of the emergence of lineages harboring some domestication signatures but that do not fit completely in the archetype of a domesticated yeast, by studying S. cerevisiae strains associated with processed olives, namely table olives, olive brine, olive oil, and alpechin. We confirmed earlier observations that reported that the Olives population results from a hybridization between S. cerevisiae and S. paradoxus. We concluded that the olive hybrids form a monophyletic lineage and that the S. cerevisiae progenitor belonged to the wine population of this species. We propose that homoploid hybridization gave rise to a diploid hybrid genome, which subsequently underwent the loss of most of the S. paradoxus sub-genome. Such a massive loss of heterozygosity was probably driven by adaptation to the new niche. We observed that olive strains are more fit to grow and survive in olive brine than control S. cerevisiae wine strains and that they appear to be adapted to cope with the presence of NaCl in olive brine through expansion of copy number of ENA genes. We also investigated whether the S. paradoxus HXT alleles retained by the Olives population were likely to contribute to the observed superior ability of these strains to consume sugars in brine. Our experiments indicate that sugar consumption profiles in the presence of NaCl are different between members of the Olives and Wine populations and only when cells are cultivated in nutritional conditions that support adaptation of their proteome to the high salt environment, which suggests that the observed differences are due to a better overall fitness of olives strains in the presence of high NaCl concentrations. Although relic olive hybrids exhibit several characteristics of a domesticated lineage, tangible benefits to humans cannot be associated with their phenotypes. These strains can be seen as a case of adaptation without positive or negative consequences to humans, that we define as a quasi-domestication.
... Sugar transporters have been extensively studied in S. cerevisiae (Luyten et al., 2002;Batista et al., 2004;Rintala et al., 2008). There are 20 identified genes in the family of hexose transporters for S. cerevisiae, of which 18 are bona fide transporters (HXT1-17 and GAL2) and two genes encode sensor proteins (SNF3 and RGT2). ...
... BLAST analysis of the scHXT7 protein against the Y. lipolytica genome revealed three of the six currently identified hexose transporters (YALI0F19184p, YALI062424p, and YALI0C09843p) with the former having highest sequence homology to HXT7. The same group showed that the rate of glucose uptake was improved with the overexpression of the GCR1 transcription factor due to its role in inducing the low-affinity HXT1 transporter thought to be critically important at the beginning of fermentation (Luyten et al., 2002). BLAST analysis for a homolog of GCR1 in Y. lipolytica yielded no matches, indicating a potentially divergent mechanism for regulation. ...
Recent advances in genetic engineering capabilities have enabled the development of oleochemical producing strains of Yarrowia lipolytica. Much of the metabolic engineering effort has focused on pathway engineering of the product using glucose as the feedstock; however, alternative substrates, including various other hexose and pentose sugars, glycerol, lipids, acetate, and less-refined carbon feedstocks, have not received the same attention. In this review, we discuss recent work leading to better utilization of alternative substrates. This review aims to provide a comprehensive understanding of the current state of knowledge for alternative substrate utilization, suggest potential pathways identified through homology in the absence of prior characterization, discuss recent work that either identifies, endogenous or cryptic metabolism, and describe metabolic engineering to improve alternative substrate utilization. Finally, we describe the critical questions and challenges that remain for engineering Y. lipolytica for better alternative substrate utilization.
... HXT2 showed low expression levels throughout fermentation. HXT3 was the most highly expressed HXT gene showing a pattern consistent with earlier results (Diderich et al. 1999;Luyten et al. 2002). Unexpectedly, HXT6/7, described as high affi nity carriers (Reifenberger et al. 1997), were expressed throughout fermentation. ...
... However, expression of HXT6 cloned from an oenological strain into a hxt1-7∆ hxt1-7∆ hxt1-7 mutant strain, could not restore growth on a medium with low glucose concentration, whereas HXT7-only cells were shown to grow only weakly on the same medium. In contrast, expression of HXT6 or HXT7 from a laboratory strain in the hxt1-7∆ hxt1-7∆ hxt1-7 mutant enabled the yeast to grow well on low glucose (Luyten et al. 2002). This result illustrates that despite high sequence homology, hexose carriers from oenological and laboratory yeast strains have dissimilar behaviours and could play different roles during wine fermentation. ...
Wine fermentation is a dynamic and complex process in which the yeast cell is subjected to multiple stress conditions. A successful adaptation involves changes in gene expression profiles where a large number of genes are up- or down-regulated. Functional genomic approaches are com- monly used to obtain global gene expression profiles, providing a comprehensive view of yeast physiology. We used Serial Analysis of Gene Expres- sion (SAGE) to quantify gene expression profiles in an industrial strain of Saccharomyces cerevisiae under simulated winemaking conditions. The transcriptome of wine yeast was analysed at three stages during fermentation of an artificial must, mid-exponential phase, and early- and late-stationary phases. Upon correlation with the yeast genome, we found three classes of transcripts: sequences which corresponded to Open Reading Frames (ORFs); expressed sequences from intergenic regions; and messengers that did not match the published reference yeast genome. In all fermentation phases studied, the most highly expressed genes related to energy production and stress response. For glycolysis related genes, different transcript levels were observed during each phase. Surprisingly, two sequential enzymes, phosphofructokinase and aldolase, showed the lowest (< 4 transcripts per cell) and the highest (up to 610 transcripts per cell) gene expression levels, respectively. Different isoenzymes, including hexose transporters (HXT), were differentially induced depending on the growth phase. About 10% of transcripts matched nonannotated-ORF regions within the yeast genome and could correspond to small novel genes originally omitted in the first gene annotation effort. Up to 22% of transcripts, particularly at late stationary phase, did not match any known location within the genome. As the available reference yeast genome was obtained from a laboratory strain, these expressed sequences could represent genes only expressed by an industrial yeast strain. Further studies are necessary to identify the role of these potential genes during wine fermentation.
... This performance possibly indicates that the hexose transporters in this evolved population acquired 358 greater affinity for fructose during the evolution, a fact that could also have been subserved by their 359 expedited ethanol tolerance. Hxt6 and Hxt7 transporters are probably the ones affected, since they are 360 essential at the final stages of the alcoholic fermentation, when the ethanol concentration in the broth 361 reaches its peak(Luyten et al. 2002). The evolved CFB population acquired in our previous study 362(Mavrommati et al. 2023) was also tested in the same alcoholic fermentation conditions. ...
Saccharomyces cerevisiae is the main yeast used in the winemaking industry. Its innate glucofilicity provokes a discrepancy in glucose and fructose consumption during alcoholic fermentation of grape must, which, combined with the inhibitory effect of ethanol accumulated in the fermentation broth, might lead to stuck or sluggish fermentations. In the present study, we realized an Adaptive Laboratory Evolution strategy, where an alcoholic fermentation of a 20 g L − 1 fructose broth was followed by cell selection in a high ethanol concentration environment, employed in two different S. cerevisiae strains, named CFB and BLR. The evolved populations originated from each strain after 100 generations of evolution exhibited diverse fermentative abilities. One evolved population, originated from CFB strain, fermented a synthetic broth of 100 g L − 1 glucose and 100 g L − 1 fructose to dryness in 170 h, whereas the parental strain did not complete the fermentation even after 1000 h of incubation. The parameters of growth of the parental and evolved populations of the present study, as well as of the ethanol tolerant populations acquired in a previous study, when grown in a synthetic broth of 100 g L − 1 glucose and 100 g L − 1 fructose, were calculated through a kinetic model and were compared to each other in order to identify the effect of evolution on the biochemical behavior of the strains. Finally, in a fermentation at synthetic broth with 200 g L − 1 fructose only the evolved population derived from CFB strain showed improved fermentative behavior than its parental strain.
... Hxt1 and Hxt3 are low-affinity transporters, Hxt6 and Hxt7 are high-affinity transporters, and Hxt2 and Hxt4 show a moderate affinity for glucose (Reifenberger et al. 1997;Buziol et al. 2008). Since yeast cells need different transporters at different growth stages, their expression changes with glucose concentration (Kruckeberg 1996;Boles and Hollenberg 1997;Luyten et al. 2002). To explore the relationship between the promotion of cell growth and glucose transport after CAT8 deletion, the transcription levels of glucose transporters Hxt1-4 and Hxt7 were measured at 24 h and 34 h by qRT-PCR according to the transcriptome analysis results. ...
Cat8 is a C6 zinc cluster transcription activator in yeast. It is generally recognized that the transcription of CAT8 is inhibited and that Cat8 is inactive in the presence of high concentrations of glucose. However, our recent study found that constitutively overexpressed Cat8 played a regulatory role in Saccharomyces cerevisiae in the presence of 20 g/L glucose. To explore the regulatory network of Cat8 at high glucose concentrations, CAT8 was both overexpressed and deleted in this study. Cell growth and glucose consumption in different media were significantly accelerated by the deletion of CAT8, while the lag period was greatly shortened. RNA-seq and genetic modification showed that the deletion of CAT8 changed the type of energy metabolism in yeast cells. Many genes related to the mitochondrial respiratory chain were downregulated, resulting in a reduction in aerobic respiration and the tricarboxylic acid cycle. Meanwhile, both the energy supply of anaerobic ethanol fermentation and the Crabtree effect of S. cerevisiae were enhanced by the deletion of CAT8. CAT8 knockout cells show a higher sugar uptake rate, a higher cell growth rate, and higher tolerance to glucose than the wild-type strain YS58. This study expands the understanding of the regulatory network of Cat8 and provides guidance for modulating yeast cell growth.
Key points
• The deletion of CAT8 promoted cell growth of S. cerevisiae.
• Transcriptome analysis revealed the regulation network of Cat8 under 1% glucose condition.
• CAT8 deletion increases the glucose tolerance of cells by enhancing the Crabtree effect.
... To identify P. striiformis f. sp. tritici hexose transporters, the protein sequences of yeast (Saccharomyces cerevisiae) hexose transporters HXT1 to HXT7 (Luyten et al. 2002) were used as queries to search against the CYR32 genome, using the tBLASTn program. Finally, 35 P. striiformis f. sp. ...
Puccinia striiformis f. sp. tritici (Pst) is the causal agent of wheat stripe rust that causes severe yield losses all over the world. As a macrocyclic heteroecious rust fungus, it is able to infect two unrelated host plants: wheat and barberry. Its urediniospores infect wheat and cause disease epidemic, while its basidiospores parasitize barberry to fulfill the sexual reproduction. This complex life cycle poses interesting questions on the different mechanisms of pathogenesis underlying the infection of the two different hosts. In the present study, transcriptomes of Pst during the initial infection of wheat and barberry leaves were qualitatively and quantitatively compared. As a result, 142 wheat-specific expressed genes (WEGs) were identified, which was far less than 2,677 barberry-specifically expressed genes (BEGs). A larger proportion of evolutionary conserved genes were observed in BEGs than that in WEGs, implying a longer history of the interaction between Pst and barberry. Additionally, Pst differentially expressed genes (DEGs) between wheat at 1 dpi/2 dpi and barberry at 3 dpi/ 4dpi were identified by quantitative analysis. Gene Ontology analysis of these DEGs and expression patterns of Pst pathogenic genes, including those encoding candidate secreted effectors, cell wall degrading enzymes, and nutrient transporters, demonstrated that urediniospores and basidiospores exploited distinct strategies to overcome host defense systems. These results represent the first analysis of the Pst transcriptome in barberry and contribute to a better understanding of the evolutionary processes and strategies of different types of rust spores during the infection process on different hosts.
... Amongst the multigenic family of 20 genes potentially implicated in hexose transport in yeast, only few have been identified as susceptible and playing a significant role in sugar transport under enological conditions. Recent results revealed that the low-affinity transporter Hxt3 plays a major role throughout wine fermentation, while the second major low-affinity transporter Hxt1 seems to be active only at the beginning of the fermentation [49]. In the same work, it has been shown that the high-affinity transporters Hxt7 and Hxt6 could play an important role in the completion of sugar utilization at the end of the fermentation, while the Hxt2 carrier is involved in growth initiation. ...
... One of the most important adaptive features of post WGD species is the capacity to consume glucose rapidly, then depleting media from nutrients, and hampering respiration in other nonfermentative cells (Gutierrez et al., 2016;Hagman & Piskur, 2015). Glucose uptake rate and metabolism directly impact CO 2 production levels, which are determined by glucose hexose transporters (encoded by HXT genes) (Luyten, Riou, & Blondin, 2002). The HXT genes have been extensively amplified in fungal lineages that have independently evolved aerobic fermentation (such as S. cerevisiae and C. glabrata), while a reduction in the number of HXT genes has been reported in aerobic respiratory species (such as K. lactis; Lin & Li, 2011), in agreement with our results. ...
The capacity of some yeasts to extract energy from single sugars, generating CO2 and ethanol (=fermentation), even in the presence of oxygen, is known as the Crabtree effect. This phenomenon represents an important adaptation as it allowed the utilization of the ecological niche given by modern fruits, an abundant source of food that emerged in the terrestrial environment in the Cretaceous. However, identifying the evolutionary events that triggered fermentative capacity in Crabtree‐positive species is challenging, as microorganisms do not leave fossil evidence. Thus, key innovations should be inferred based only on traits measured under culture conditions. Here, we reanalyzed data from a common garden experiment where several proxies of fermentative capacity were recorded in Crabtree‐positive and Crabtree‐negative species, representing yeast phylogenetic diversity. In particular, we applied the “lasso‐OU” algorithm which detects points of adaptive shifts, using traits that are proxies of fermentative performance. We tested whether multiple events or a single event explains the actual fermentative capacity of yeasts. According to the lasso‐OU procedure, evolutionary changes in the three proxies of fermentative capacity that we considered (i.e., glycerol production, ethanol yield, and respiratory quotient) are consistent with a single evolutionary episode (a whole‐genomic duplication, WGD), instead of a series of small genomic rearrangements. Thus, the WGD appears as the key event behind the diversification of fermentative yeasts, which by increasing gene dosage, and maximized their capacity of energy extraction for exploiting the new ecological niche provided by single sugars. Whole‐genomic duplication and fermentative capacity in yeast evolution.
... transporter, transfer/carrier, transcription factors, signalling molecule and defence. Luyten et al. (2002), Henricsson et al. (2005) and Young et al. (2011) reported that transporter proteins that play a pivotal role during the transport of molecules (e.g. hexose sugars) from the grape must into the cell are overexpressed during the lag phase, as the cells are adapting to their environment prior to active proliferation. ...
... This could be due to absence of residual RS that was a source for competitive mono and disaccharides that block the transportation of glucose into yeast cells. This was clarified by Kattie et al., (2002) who mentioned the competitive action of hexoses on transportation of glucose into yeast and Rosario (1993) who added that both galactose and glucose competed on the same port responsible for transportation of glucose into the yeast cell. On the other hand, Patrick (2006) clarified the role of oligosaccharides specially sucrose in fermentation by yeast and its ability to be transported to inside. ...
Successive saccharification and fermentation was to evaluate fermentation by Saccharomyces cerevisiae and/or Phanerochaete chrysosporium using the pre-saccharified rice straw (RS) compared to glucose source as control. P. chrysosporium cellulases produced on carboxymethyl cellulose (CMC) or pretreated RS were used in saccharifying new pretreated RS into fermentable sugars (4.5-5.5%). The superiority of S. cerevisiae in fermenting pure glucose and RS was observed in all cases. Antagonism was absent between yeast and fungi during fermentation of pure glucose, as 42 g ethanol/100g glucose was produced when yeast was used individually or in mixed culture with the fungus. Nevertheless, ethanol production on the non-sterilized RS (saccharified with rice straw cellulases source) was positively correlated with fungal presence, as it reached 28.24 and 24.18 g ethanol/100g glucose, on the other hand sterilization decreased ethanol production to 16.20 and 20.1 g ethanol/ 100 g glucose, when yeast was used individually or mixed with fungi, respectively. There was no difference in production magnitude if correlated to cellulases source substrate whether it was rice straw or CMC.
... transporter, transfer/carrier, transcription factors, signalling molecule and defence. Luyten et al. (2002), Henricsson et al. (2005) and Young et al. (2011) reported that transporter proteins that play a pivotal role during the transport of molecules (e.g. hexose sugars) from the grape must into the cell are overexpressed during the lag phase, as the cells are adapting to their environment prior to active proliferation. ...
A single Saccharomyces cerevisiae wine yeast strain produces a range of aroma and flavour metabolites
(e.g. volatile thiols), as well as unfavourable metabolites (e.g. volatile acidity [VA]), during the alcoholic
fermentation of white wine, especially Sauvignon blanc. The former contribute to the organoleptic
quality of the final wine. Previous research showed that yeast-derived enzymes (proteins) are involved in
the release of wine quality-enhancing or quality-reducing metabolites during fermentation. Small-scale
winemaking trials were initiated to evaluate the protein expression and metabolite release of S. cerevisiae
hybrid yeasts producing tropical fruit aroma. Commercial ‘thiol-releasing’ wine yeasts (TRWY) were
included in winemaking trials as references. Improved hybrids were identified that showed enhanced thiolreleasing
abilities, specifically 3-mercaptohexanol (3MH), and lower VA formation during the production
of Sauvignon blanc wines compared to some commercial TRWY references. It is noteworthy that the
hybrid NH 56 produced wines with the second highest 3MH levels after hybrid NH 84, and with the lowest
acetic acid of all strains included in this study. This yeast was also the only strain to have downregulated
proteins linked to amino acid biosynthesis, the pentose phosphate pathway, glycolysis, and fructose and
galactose metabolism during the lag phase. Furthermore, differences in protein expression were reflected
in the variation in metabolite release by different strains, thereby confirming that enzymes (proteins) are
the final effectors of metabolite release.
... Therefore, the research of several laboratories focus on phosphorylation by the HXK1-and HXK2-encoded hexokinases and the GLK1-encoded glucokinase, as well as on hexose transporters encoded by HXT1-HXT18 and SNF3. The low affinity hexose transporter Hxt3p and high affinity transporters Hxt6p and Hxt7p play particularly important roles in wine fermentation (75). It is possible that increased expression of these transporters will decrease the occurrence of stuck fermentation. ...
It is the nature of change that big events erupt suddenly and noisily, grab the headlines and shake the world. Changes that often have more impact on mankind’s longterm future tend to take place much more slowly and quietly. It is in our nature to pay too much attention to short-term events and tentative viewpoints; larger, seismic shifts happen on a scale too great for us to easily bring into focus. Futurists concur that the world at the start of the third millennium is on the verge of an era of massive and unprecedented change, change so dynamic and far-reaching as to make many facets of society virtually unrecognisable during the next few decades. We are already starting to feel tremors from what will be tectonic transformations. In this context, we might remind ourselves of the adage ‘the future belongs to those who prepare for it.’
... S. cerevisiae encodes a total of 17 putative low-, moderate-and high-affinity hexose transporters, Hxt1p to Hxt17p, the galactose transporter Gal2p and two glucose sensors, Snf3p and Rgt2p (Kruckeberg, 1996;Luyten et al., 2002;Horák, 2013). The Hxtp transporters are closely related proteins that contain 12 putative membrane-spanning domains and mediate the transport of hexoses across the cytoplasmic membrane by the mechanism of facilitated diffusion. ...
The transport of sugars across the plasma membrane is a critical step in the utilization of glucose and fructose by Saccharomyces cerevisiae during must fermentations. Variations in the molecular structure of hexose transporters and kinases may affect the ability of wine yeast strains to finish sugar fermentation even under stressful wine conditions. In this context, we sequenced and compared genes encoding the hexose transporter Hxt3p and the kinases Hxk1p/Hxk2p of Saccharomyces strains and interspecies hybrids with different industrial usage and regional background. The Hxt3p primary structure varied in a small set of amino acids, which characterized robust yeast strains used for the production of sparkling wine or to restart stuck fermentations. In addition, interspecies hybrid strains, previously isolated at the end of spontaneous fermentations, revealed a common amino acid signature. The location and potential influence of the amino acids exchanges is discussed by means of a first modelled Hxt3p structure. In comparison, hexokinase genes were more conserved in different Saccharomyces strains and hybrids. Thus, molecular variants of the hexose carrier Hxt3p, but not of kinases correlate with different fermentation performances of yeast.
Copyright © 2015 John Wiley & Sons, Ltd.
... Amongst the multigenic family of 20 genes potentially implicated in hexose transport in yeast, only few have been identified as susceptible and playing a significant role in sugar transport under enological conditions. Recent results revealed that the low-affinity transporter Hxt3 plays a major role throughout wine fermentation, while the second major low-affinity transporter Hxt1 seems to be active only at the beginning of the fermentation [49]. In the same work, it has been shown that the high-affinity transporters Hxt7 and Hxt6 could play an important role in the completion of sugar utilization at the end of the fermentation, while the Hxt2 carrier is involved in growth initiation. ...
... Individual transporters have specific functions, since they all possess different substrate affinities or specificities such as (i) low-affinity Hxt1p and Hxt3p [Km(glucose) 100 mM]; (ii) moderate to low affinity Hxt2p and Hxt4p [Km(glucose), 10 mM]; and (iii) high affinity Hxt6p and Hxt7p [Km(glucose) 1-2 mM] [24]. Differences in individual HXT gene expression are not only dependent upon the concentration of available glucose but also upon osmotic pressure, starvation, and the physiological state of the cell [1,15,16,[25][26][27][28][29][30][31][32]. ...
To characterize the mechanisms involved in glucose transport, in the filamentous fungus Aspergillus nidulans, we have identified four glucose transporter encoding genes hxtB-E. We evaluated the ability of hxtB-E to functionally complement the Saccharomyces cerevisiae EBY.VW4000 strain that is unable to grow on glucose, fructose, mannose or galactose as single carbon source. In S. cerevisiae HxtB-E were targeted to the plasma membrane. The expression of HxtB, HxtC and HxtE was able to restore growth on glucose, fructose, mannose or galactose, indicating that these transporters accept multiple sugars as a substrate through an energy dependent process. A tenfold excess of unlabeled maltose, galactose, fructose, and mannose were able to inhibit glucose uptake to different levels (50 to 80 %) in these s. cerevisiae complemented strains. Moreover, experiments with cyanide-m-chlorophenylhydrazone (CCCP), strongly suggest that hxtB, -C, and -E mediate glucose transport via active proton symport. The A. nidulans ΔhxtB, ΔhxtC or ΔhxtE null mutants showed ~2.5-fold reduction in the affinity for glucose, while ΔhxtB and -C also showed a 2-fold reduction in the capacity for glucose uptake. The ΔhxtD mutant had a 7.8-fold reduction in affinity, but a 3-fold increase in the capacity for glucose uptake. However, only the ΔhxtB mutant strain showed a detectable decreased rate of glucose consumption at low concentrations and an increased resistance to 2-deoxyglucose.
... Knowledge on yeast sugar transporters regards mostly glucose permeases. There is considerable information accumulated on their genes and regulation (Diderich et al., 1999; Luyten et al., 2002; Verwaal et al., 2004), while their physiology/biochemistry characterization remains infrequent (Hamacher et al., 2002; Lee et al., 2002 ) and controversial (for a review, see Jansen et al., 2002; Conde et al., 2010). This is partly due to the fact that yeasts consume glucose preferably to any other carbon source. ...
The closely related yeasts Debaryomyces fabryi and Debaryomyces hansenii are excellent xylose consumers. We previously described the activity of a high affinity xylose/H(+) symport from an industrial strain of D. hansenii subsequently re-classified as D. fabryi. We now report the identification of the gene encoding this permease, AY347871.2. This was retrieved from D. fabryi gDNA using a degenerate primer-PCR strategy, based on conserved regions from the amino acid sequences of three well-characterised bacterial xylose/H(+) symporters. This sequence is 86% identical to another, DEHA2C11374p from D. hansenii type strain. DEHA2C11374p was conceptually ascribed to the Major Facilitator Superfamily. The putative amino acid sequence of AY347871.2 and DEHA2C11374p presented a hydrophobicity pattern compatible with plasma membrane proteins. The last was functionally expressed in Saccharomyces cerevisiae. The sensitivity of transport activity to a protonophore, confirmed its dependence on proton motive force, as expected from a symporter. We named D. fabryi AY347871.2 and D. hansenii DEHA2C11374p as XYLH from Xylose/H(+) symport. Based on the very high similarity, we suggested that Scheffersomyces stipitis Xut3 and Aspergilus nidulans AN8400.2 may also encode xylose high affinity permeases. This article is protected by copyright. All rights reserved.
... The Hxt2 transporter has been shown to exist in forms with differing affinity for substrate [28] although under some conditions it appears to be a simple high affinity transporter [29]. Hxt2p has also been shown to be expressed early in wine fermentation when sugar substrates are in high concentration, but then is quickly degraded [30][31][32]. These observations suggest that the HXT2 protein may be an important transporter for the transition between substrate concentrations and that the affinity of the transporter may be impacted by other factors within the cell. ...
Background
The SNF3 gene in the yeast Saccharomyces cerevisiae encodes a low glucose sensor that regulates expression of an important subset of the hexose transporter (HXT) superfamily. Null mutations of snf3 result in a defect in growth on low glucose concentrations due to the inability to relieve repression of a subset of the HXT genes. The snf3 null mutation phenotype is suppressed by the loss of either one of the downstream co-repressor proteins Rgt1p or Mth1p. The relief of repression allows expression of HXT transporter proteins, the resumption of glucose uptake and therefore of growth in the absence of a functional Snf3 sensor.
Results
Strains heterozygous for both the RGT1 and MTH1 genes (RGT1/rgt1Δ MTH1/mth1Δ snf3Δ/snf3Δ) but homozygous for the snf3∆ were found to grow on low glucose. Since null alleles in the heterozygous state lead to suppression, MTH1 and RGT1 display the phenomenon of combined haploinsufficiency. This observed haploinsufficiency is consistent with the finding of repressor titration as a mechanism of suppression of snf3. Mutants of the STD1 homolog of MTH1 did not display haploinsufficiency singly or in combination with mutations in RGT1. HXT gene reporter fusion assays indicated that the presence of heterozygosity at the MTH1 and RGT1 alleles leads to increased expression of the HXT2 gene. Deletion of the HXT2 gene in a heterozygous diploid, RGT1/rgt1Δ MTH1/mth1Δ snf3Δ/snf3Δ hxt2Δ/hxt2Δ, prevented the suppression of snf3Δ.
Conclusions
These findings support the model of relief of repression as the mechanism of restoration of growth on low glucose concentrations in the absence of functional Snf3p. Further, the observation that HXT2 is the gene responsible for restoration of growth under these conditions suggests that the numbers of repressor binding domains found in the regulatory regions of members of the HXT family may have biological relevance and enable differential regulation.
... Most of the available information concerning S. cerevisiae genes was obtained under laboratory conditions, without taking into account the specific physiological situation faced by yeasts in wine-making. Various functional genomic approaches have been applied to wine yeast to specify the role of genes playing a key role in alcoholic fermentation conditions, such as the ALD or HXT genes (Saint-Prix et al. 2004;Luyten et al. 2002). Genome-wide approaches have been used to investigate gene expression patterns under enologically relevant situa- ...
Saccharomyces cerevisiae and related yeasts play a major role in wine alcoholic fermentations. These yeasts have genetic and physiological properties distinct from they laboratory counterparts. These properties are supported by genome variations that include alteration of the S. cerevisiae genome as well as the generation of hybrid genomes. Investigation of the architecture of Saccharomyces wine yeast genome have revealed important structural variations, some reflecting clearly the adaptation to the harsh wine environment. The genomic diversity results from exploitation of the many possible mechanisms underlying genome plasticity in yeast, including changes in ploidy, chromosomal translocations, nucleotides polymorphisms.The availability of new genome scanning technologies have greatly enhanced our knowledge on yeast genome and opened new opportunities to uncover the genetic basis of their technological properties.
... In the first place, the efficient utilisation of grape sugar (glucose and fructose) by S. cerevisiae and a rapid rate of glycolytic flux relies on the presence of functional alleles of genes encoding the key glycolytic enzymes, hexokinase, glucokinase, phosphoglucose isomerase, phosphofructokinase, aldolase, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, pyruvate decarboxylase and alcohol dehydrogenase (Pretorius 2000). Secondly, the low-affinity hexose transporter Hxt3p and high-affinity transporters Hxt6p and Hxt7p play particularly important roles in wine fermentation (Luyten et al. 2002). Since wine yeast strains are glucophilic, it is possible that increased expression of these S. cerevisiae transporters together with fructosespecific transporters from fructophilic yeasts (e.g. ...
The image of wine as a harmonious blend of nature, art and science invites tension between tradition and innovation, and no tension in the business of making wine is greater than that brought into play by the potential afforded by 21st century grape and wine biotechnology. The challenge is to realise the potential of technological innovation without stripping the ancient art of grapegrowing and winemaking of its charm, mysticism and romanticism. Equally challenging is the multitude of complex and interconnected agronomic, business, regulatory and social obstacles currently blocking commercial availability of transgenic grapes, wine yeast and malolactic bacterial starter strains. While the need to assess rigorously the potential negative impacts of new technologies is self-evident, over the long term, failure to overcome these hurdles will disadvantage the international wine sector and consumers alike. This contention is illustrated with reference to recent examples of genetically improved grapevine, yeast and bacterial prototypes showing potential for enhanced, cost-effective production of wine with minimised resource inputs, improved quality and low environmental impact.
... Increasing sugar uptake through the general hexose transporters, however, is more complicated. This is because some of these have specific roles during fermentation; in fact overexpression of a single HXT gene cannot fully restore the fermentation capacity of an HXT null strain (Luyten et al. 2002). In order to obtain increased general hexose uptake, it may be necessary to co-overexpress several members of the HXT gene family. ...
... The efficiency of the uptake of sugars is altered by the gene expression, where a set of specific hexose transporters (HXT) are expressed in response to environmental sig- nals [73,74] . Moreover, these transporters show competitive inhibition between glucose and fructose, and their activity is modulated by temperature through changes in the activation energy of the process as well as by noncompetitive inhibition by ethanol [75] . Kinetic expressions that considered all these effects were incorporated as UB (upper bound) into the idFV715 model (Additional file 2: eqs. 1, 2). ...
Yeast is considered to be a workhorse of the biotechnology industry for the production of many value-added chemicals, alcoholic beverages and biofuels. Optimization of the fermentation is a challenging task that greatly benefits from dynamic models able to accurately describe and predict the fermentation profile and resulting products under different genetic and environmental conditions. In this article, we developed and validated a genome-scale dynamic flux balance model, using experimentally determined kinetic constraints.
Appropriate equations for maintenance, biomass composition, anaerobic metabolism and nutrient uptake are key to improve model performance, especially for predicting glycerol and ethanol synthesis. Prediction profiles of synthesis and consumption of the main metabolites involved in alcoholic fermentation closely agreed with experimental data obtained from numerous lab and industrial fermentations under different environmental conditions. Finally, fermentation simulations of genetically engineered yeasts closely reproduced previously reported experimental results regarding final concentrations of the main fermentation products such as ethanol and glycerol.
A useful tool to describe, understand and predict metabolite production in batch yeast cultures was developed. The resulting model, if used wisely, could help to search for new metabolic engineering strategies to manage ethanol content in batch fermentations.
... However, with the caveat that our study examined sugar utilisation rather than growth, the mismatch we observed between a glucose and fructose mixture and that of fructose alone (R 2 = 0.083) suggests it unlikely that a fructose-only medium will be useful to screen for performance in an extended mixed sugar fermentation. The presence of glucose is highly inXuential on fructose metabolism [14,18,19,21,27,28,32,37,38]. At this point it is not possible to state how glucose inXuenced fructose consumption. ...
The yeast Saccharomyces cerevisiae has a fundamental role in fermenting grape juice to wine. During alcoholic fermentation its catabolic activity converts sugars (which in grape juice are a near equal ratio of glucose and fructose) and other grape compounds into ethanol, carbon dioxide and sensorily important metabolites. However, S. cerevisiae typically utilises glucose and fructose with different efficiency: glucose is preferred and is consumed at a higher rate than fructose. This results in an increasing difference between the concentrations of glucose and fructose during fermentation. In this study 20 commercially available strains were investigated to determine their relative abilities to utilise glucose and fructose. Parameters measured included fermentation duration and the kinetics of utilisation of fructose when supplied as sole carbon source or in an equimolar mix with glucose. The data were then analysed using mathematical calculations in an effort to identify fermentation attributes which were indicative of overall fructose utilisation and fermentation performance. Fermentation durations ranged from 74.6 to over 150 h, with clear differences in the degree to which glucose utilisation was preferential. Given this variability we sought to gain a more holistic indication of strain performance that was independent of fermentation rate and therefore utilized the area under the curve (AUC) of fermentation of individual or combined sugars. In this way it was possible to rank the 20 strains for their ability to consume fructose relative to glucose. Moreover, it was shown that fermentations performed in media containing fructose as sole carbon source did not predict the fructophilicity of strains in wine-like conditions (equimolar mixture of glucose and fructose). This work provides important information for programs which seek to generate strains that are faster or more reliable fermenters.
... The possible explanation for this phenotype could be as follows. In a recent study by Luyten et al. (2002), it has been shown that Hxt3p has the highest capacity to support fermentation on fructose. That means, since HXT3 is expressed in hxt1 strain, it consumed fructose at higher rates and produced very high ethanol when compared to hxt3 strain. ...
A novel method for high-throughput stoichiometric and metabolic flux profiling was developed and a set of deletion mutants of S. cerevisiae, which are known to be involved in central carbon metabolism were selected and investigated on glucose, galactose and fructose. On glucose and fructose, the growth was predominantly fermentative and on galactose, respiration was more active. mae1D strain did not show any significant growth phenotype on glucose, however, it had highest PPP flux on galactose, which could be due to redirection of NADPH production to the PPP. On fructose, mae1D strain had highest oxygen uptake rate with very low ethanol yield, which could be due to reduced PPP flux and to maintain NADPH levels either via NADPH specific -isocitrate dehydrogenase or -aldehyde dehydro-genase. imp2'D strain had lowest PPP flux and very high respiratory activity on galactose; and pck1D strain had lowest PPP flux on glucose, which might also point to a possible activation of malic enzyme. On fructose, hxt17D strain had highest sugar consumption and ethanol production rates and imp2'D strain had highest ethanol yield. The functional prediction of hypothetical genes by utilising this quantitative data using computational analyses suggested a possible role in glycolysis or pyruvate metabolism for YBR184W and low affinity transporter role for YIL170W.
... The apparent K m value was lower (about 2.4 mM) in anaerobically growing rho 0 cells (data not shown), in which a high level of endogenous oxygen consumption could be detected. Therefore, in glucoselimiting concentrations, the oxygen consumption activity of both strains may depend on: (a) the activity of the hexose transporter (Luyten et al., 2002); (b) the activities of hexokinase and of glucose kinase; and/or (c) other steps involved in the control of glucose metabolic pathways. Moreover, neither monocarboxylic acids (DL-lactate, pyruvate or acetate, which easily enter cells at pH 3.3), acetaldehyde, ethanol nor glycerol affected endogenous and glucose-dependent oxygen fluxes (data not shown). ...
Despite the absence of an alternative mitochondrial ubiquinol oxidase, Saccharomyces cerevisiae consumes oxygen in an antimycin A- and cyanide-resistant manner. Cyanide-resistant respiration is typically used when the classical respiratory chain is impaired or absent (i.e in anaerobically-grown cells shifted to normoxia or in respiratory-deficient cells). We characterized the non-respiratory oxygen consumption pathways operating during anoxic-normoxic transitions in glucose-repressed resting cells. High-resolution oxygraphy confirmed that the cellular non-respiratory oxygen consumption pathway is sensitive to high concentrations of cyanide, azide, SHAM and TTFA, and revealed several new characteristics. First, the use of sterol biosynthesis inhibitors showed that this pathway makes a considerable contribution (about 25%) to both endogenous and glucose-dependent oxygen consumption. Anaerobically-grown glucose-repressed cells exhibited high apparent oxygen affinities (K(m) for oxygen = 0.5-1 micro M), even in mutants deficient in respiration or sterol synthesis. Exogeneously added glucose and endogenous stored carbohydrates were the only substrates that were efficient for cellular oxygen consumption (apparent K(m) for exogenous glucose = 2-3 mM). On the other hand, fluorimetric measurements of the cellular NAD(P)H pool showed that the cellular oxygen consumption (sterol biosynthesis and unknown pathways) was dependent more on the intracellular level of NADPH than of NADH. High oxygen affinity NADPH-dependent oxygen consumption systems were thought to be mainly localized in microsomal membranes, and several data indicated a significant contribution made by uncoupled p450 systems, together with still uncharacterized systems. Such activities are associated in vitro with a massive production of O(2) (.-) and, to a lower extent, H(2)O(2) and a likely concomitant production of H(2)O.
... S. cerevisiae has to carry out fermentative metabolism when glucose is available at a high concentration (grape must). Fermentation is a process that yields low amounts of energy (2 mol of ATP/mol of glucose), so a high flux of glucose, transported by a number of permeases, is necessary to ensure the generation of enough energy during processes such as enological fermentation, when sugar availability changes constantly (25). Environments inhabited by Trichoderma strains are nutrient poor, and their resources are exploited by their extracellular hydrolases. ...
Using a differential display technique, the gene gtt1, which codes for a high-affinity glucose transporter, has been cloned from the mycoparasite fungus Trichoderma harzianum CECT 2413. The deduced protein sequence of the gtt1 gene shows the 12 transmembrane domains typical of sugar transporters, together with certain residues involved in glucose
uptake, such as a conserved arginine between domains IV and V and an aromatic residue (Phe) in the sequence of domain X. The
gtt1 gene is transcriptionally regulated, being repressed at high levels of glucose. When carbon sources other than glucose are
utilized, gtt1 repression is partially alleviated. Full derepression of gtt1 is obtained when the fungus is grown in the presence of low carbon source concentrations. This regulation pattern correlates
with the role of this gene in glucose uptake during carbon starvation. Gene expression is also controlled by pH, so that the
gtt1 gene is repressed at pH 6 but not at pH 3, a fact which represents a novel aspect of the influence of pH on the gene expression
of transporters. pH also affects glucose transport, since a strongly acidic pH provokes a 40% decrease in glucose transport
velocity. Biochemical characterization of the transport shows a very low Km value for glucose (12 μM). A transformant strain that overexpresses the gtt1 gene shows a threefold increase in glucose but not galactose or xylose uptake, a finding which confirms the role of the gtt1 gene in glucose transport. The cloning of the first filamentous ascomycete glucose transporter is the first step in elucidating
the mechanisms of glucose uptake and carbon repression in aerobic fungi.
Wine yeast have been exposed to harsh conditions for millennia, which have led to adaptive evolutionary strategies. Thus, wine yeasts from Saccharomyces genus are considered an interesting and highly valuable model to study human-drive domestication processes. The rise of whole-genome sequencing technologies together with new long reads platforms has provided new understanding about the population structure and the evolution of wine yeasts. Population genomics studies have indicated domestication fingerprints in wine yeast, including nucleotide variations, chromosomal rearrangements, horizontal gene transfer or hybridization, among others. These genetic changes contribute to genetically and phenotypically distinct strains. This review will summarize and discuss recent research on evolutionary trajectories of wine yeasts, highlighting the domestication hallmarks identified in this group of yeast.
The objective of this study was to determine glucose isomerase activity in different prepared original or synthetic wine media to prevent sluggish or stuck fermentation, which may be caused by sugar uptake deficiency in yeast. The unfermented grape juice contains almost equal amounts of glucose and fructose. After fermentation, the residual sugar is mostly fructose, this is called glucose/fructose discrepancy (GFD) and is caused by the affinity decrease of hexose transporters towards fructose as ethanol accumulates. This results in stuck fermentation and is unwanted as the wine is sweet and risks microbial spoilage. Converting remaining fructose to glucose by glucose isomerase may be a solution so we tested the activity of this enzyme in synthetic and original wine media. Glucose formation, 0.5 % w/v, from 1% w/v fructose took place in synthetic wine medium containing 13 % v/v ethanol, 1% w/v glycerol and at pH 3.3. In original wine medium glucose formation did not take place except when wine was diluted at least five folds and at pH values equal or higher than 6 whether if tartaric acid was present or not. Since neither dilution, nor pH adjustment can be applicable, other ways to employ this enzyme should be tried.
Yarrowia lipolytica is a safe and robust yeast to efficiently use lipid as the sole carbon source, which provides us opportunities for biomanufacturing of a series of high-value products from cost-effective agriculture feedstocks such as plant oils and animal fats. Y. lipolytica has a unique propensity for high flux through tricarboxylic acid (TCA) cycle intermediates and biological precursors such as acetyl-CoA and malonyl-CoA that can be diverted into a variety of heterologous value-added bioproducts. With recent advances in metabolic engineering and synthetic biology tools, the potential of using Y. lipolytica for biomanufacturing of high-value products has been expanded. Examples include industrial enzymes, extracellular proteins, fatty alcohols, wax esters, long-chain diacids, omega-3 fatty acids, and carotenoids. For large-scale biomanufacturing using oils/fats as substrate, the poor mixing and mass transfer caused by the insolubility of substrates in an aqueous medium is one of the major challenges that have to be addressed in addition to the pathway engineering and optimization for both fatty acid biosynthesis and conversion. The multi-phase computational fluid dynamics (CFD) simulation can be used as a powerful tool for analysis of mixing and mass transfer behaviors in bioreactors and further guide the bioreactor design and optimization of operating conditions. Cell morphology has a profound effect on cell growth, oil substrate uptake, and product formation. Both PKA and cAMP-dependent signaling pathways are involved in the dimorphic transition in Y. lipolytica. Maintaining the dimorphic yeast shape via morphology engineering strategies has been explored. This chapter also introduced several examples of how we combined cell morphology engineering, metabolic pathway optimization, and bioreaction engineering to significantly improve the production of intracellular lipids, citric acid, and wax esters from plant oils. Potential strategies for further improving the biosynthesis efficiency via transporter engineering in yeast were also introduced.KeywordsBiomanufacturingMetabolic engineeringCell morphologyCFD simulationOils and fats
Yarrowia lipolytica
Patulin is a mycotoxin produced by Penicillium expansum, that exists in apple and cider, and threatens human health and food safety. Saccharomyces cerevisiae (S. cerevisiae) can inhibit and degrade patulin during cider fermentation. Nonetheless, the specific patulin metabolic mechanism is still unclear. Thus, Illumina RNA-Seq technology was applied in this study to analyze the transcriptome of S. cerevisiae cultured in medium with and without patulin, and the gene expression studied. The results showed that the genes encoding redox reactions (OYE3, GTT2, GPX2), cell-to-drug transmembrane transport (HXT, FLR1, YCF1), and responses to biological stress (Yap1, Msn1) were significantly upregulated. S. cerevisiae can respond to the stress of patulin by increasing the activity of related resistant enzymes, such as aryl-alcohol dehydrogenase, NADPH dehydrogenase 3, and glutathione S-transferase 2, which leads to effective degradation of patulin. The findings of this study explore the degradation mechanism of patulin and provide potential biomarker genes for detecting patulin in cider.
Alkaline pretreatment is an efficient method to destroy the lignocellulose structure of rice straw for bioethanol production. This process generates various toxic compounds, such as ferulic acid, which could inhibit bioethanol production. In this study, a waste biomass-based adsorbent from the enzymatic hydrolysis of rice straw (AEPA250) was used for ferulic acid detoxification in the alkali-pretreated hydrolysate. The AEPA250 detoxification process was first optimized by response surface methodology to increase ferulic acid removal, while reducing glucose loss. This optimization of detoxification resulted in 99.268% ferulic acid removal coupled with 3.028% glucose loss. The fermentation processing parameters and bioethanol production for different fermentation systems were evaluated after adopting the optimal AEPA250 detoxification conditions to select the most suitable optimization method. The result revealed that optimized detoxified fermentation with AEPA250 filtration (ODF) system was more suitable for bioethanol production than optimized detoxified fermentation containing AEPA250 (ODFA) system followed by non-detoxified fermentation (NDF) system. A logistic model was applied to further compare the yeast growth kinetics of the ODF and NDF systems. The ODF system had a maximum specific growth rate (μmax) of 0.532 h⁻¹, indicating that the ODF system possessed greater bioethanol fermentation potential. Metabonomics and transcriptomics analyses were used to identify differentially expressed metabolites and functional genes that contribute to detoxification by the ODF system. AEPA250 was produced by the enzymatic hydrolysis of biomass waste of rice straw in the bioethanol production process, and a self-sufficient bioethanol production system could be established by in-situ AEPA250 detoxification using the ODF system. These results pave the way for the realization of a cleaner bioethanol production process by reducing environmental waste production, exogenous adsorbent use, and the cost of enzymatic hydrolysis residue treatment.
The predominant feature in wine-making is the conversion of sugars contained in grape mashes or musts into ethanol, a task almost exclusively fulfilled by unicellular eukaryotes which divide by budding—the yeasts. Whereas several non-Saccharomyces yeast species are present in the early stages of fermentation, Saccharomyces cerevisiae commonly added in starter cultures generally outgrows all other yeasts in the process of vinification and determines the principle quality of the final product. In the first part of this chapter we recapitulate the pathway of alcoholic fermentation, with special focus on the physiological properties relevant in vinification and the molecular differences discovered between laboratory strains of S. cerevisiae and their relatives employed in wine production. We present the current view on how hexose transport and hexose phosphorylation are especially adapted to the environments encountered during wine production. The generation of organoleptic important by-products of alcoholic fermentation, such as glycerol and acetate, are also discussed. Finally, the three major signaling pathways governing the response to sugar availability, SNF1, cAMP/Ras, and Snf3-Rgt1/2, are briefly explained with relation to wine yeast. An outlook on the growing importance of non-Saccharomyces yeasts and the expected impact of modern high-throughput techniques concludes this review.
The yeast Saccharomyces cerevisiae has evolved to dominate grape juice fermentation. A suite of cellular properties, rapid nutrient depletion, production of inhibitory compounds and the metabolic narrowing of the niche, all enable a minor resident of the initial population to dramatically increase its relative biomass in the ecosystem. This dominance of the grape juice environment is fueled by a rapid launch of glycolysis and energy generation mediated by transport of hexoses and an efficient coupling of transport and catabolism. Fermentation occurs in the presence of molecular oxygen as the choice between respiratory or fermentative growth is regulated by the availability of sugar a phenomenon known as glucose or catabolite repression. Induction of the [GAR⁺] prion alters the expression of the major hexose transporter active under these conditions, Hxt3, reducing glycolytic capacity. Bacteria present in the grape juice ecosystem were able to induce the [GAR⁺] prion in wine strains of S. cerevisiae. This induction reduced fermentation capacity but did not block it entirely. However, dominance factors such as the rapid depletion of amino acids and other nitrogen sources from the environment were impeded enabling greater access to these substrates for the bacteria. Bacteria associated with arrested commercial wine fermentations were able to induce the prion state, and yeast cells isolated from arrested commercial fermentations were found to be [GAR⁺] thus confirming the ecological relevance of prion induction. Subsequent analyses demonstrated that the presence of environmental acetic acid could lead to [GAR⁺] induction in yeast strains under certain conditions. The induction of the prion enabled yeast growth on non-preferred substrates, oxidation and reduction products of glucose and fructose, present as a consequence of bacterial energy production. In native ecosystems prion induction never exceeded roughly 50–60% of the population of yeast cells suggesting that the population retains the capacity for maximal fermentation. Thus, the bacterial induction of the [GAR⁺] prion represents a novel environmental response: the query of the environment for the presence of competing organisms and the biological decision to temper glucose repression and dominance and enter a metabolic state enabling coexistence.
Wine plays a major role in the economies of many nations. The world’s annual wine production from about 8 million hectares of vineyards totals around 27 billion litres (1). Shifting consumer preferences, globalisation and other economic factors have forced an evolution of the international wine industry from a “cottage industry” of independent producers to global networks of consumer-conscious enterprises (2,3).
In Saccharomyces cerevisiae the process of transport of sugar substrates into the cell comprises a complex network of transporters and interacting regulatory mechanisms. Members of the large family of hexose (HXT) transporters display uptake efficiencies consistent with their environmental expression and play physiological roles in addition to feeding the glycolytic pathway. Multiple glucose-inducing and glucose-independent mechanisms serve to regulate expression of the sugar transporters in yeast assuring that expression levels and transporter activity are coordinated with cellular metabolism and energy needs. The expression of sugar transport activity is modulated by other nutritional and environmental factors that may override glucose-generated signals. Transporter expression and activity is regulated transcriptionally, post-transcriptionally and post-translationally. Recent studies have expanded upon this suite of regulatory mechanisms to include transcriptional expression fine tuning mediated by antisense RNA and prion-based regulation of transcription. Much remains to be learned about cell biology from the continued analysis of this dynamic process of substrate acquisition.
Objective The changes in the gene expression profile of Candida albicans associated with the acquisition of experimentally induced resistance to terbinafine was studied by genomic expression profiling. Methods C. albicans ATCC 90028 was passed in increasing concentrations of terbinafine to generate isolate ATCC 90028-R. Susceptibility testing revealed that ATCC 90028-R was highly resistant to terbinafine. The gene expression profile of ATCC 90028-R was compared with that of ATCC 90028 using DNA microarray analysis. Results Upon examination of MICs of antifungal compounds, it was found that ATCC 90028-R was resistant to terbinafine. By DNA microarray analysis, a total of 109 genes were found to be differentially expressed, 46 genes were up-regulated in ATCC 90028-R, while 63 genes down-regulated by at least 100%. Conclusions The results suggest that the genes associated with the induced molecule transporter and detoxification, repressed protein synthesis, energy generation, sugar transporter, cell stress, and ionic homeostasis may contribute to terbinafine resistance.
During aerobic growth at high glucose concentrations, the yeast Saccharomyces cerevisiae prefers to catabolise glucose through fermentation rather than through respiration. Previously fully respiratory growth could only be achieved when a low external glucose concentration was maintained, in fed-batch and continuous cultures, but since the development of the respiratory strain, TM6*, in our laboratory this can now be achieved even at high external glucose concentration. In this thesis the transferability of the respiratory phenotype has been confirmed by integration of the chimeric TM6* construct into a hxt1-hxt7 wine strain. The genomic profile of the resulting V5.TM6*P strain was investigated by miniarray and the results showed that genes involved in the TCA cycle, glyoxylate cycle, gluconeogenesis pathway and the respiratory chain were upregulated compared to the parental strain. The transcriptional responses in the respiratory strain were not altered when the external glucose concentration changed in contrast to the wild type strain where a change in transcription (both up and down regulated) was observed when glucose was changed from high to low concentration in a batch culture. It was further observed that the respiratory strain had a lower concentration of fructose-1, 6-bisphosphate compared to its parent strain. Modelling of regulation of glycolysis showed that there are differences in the kinetics of the phosphofructokinase between the TM6* strain and the wild type; suggesting a key role of this enzyme in the shift between a respiro-fermentative and a respiratory metabolism. As a result of its respiratory metabolism, the TM6* strain is able to produce more biomass, making the TM6* strain an excellent candidate as a protein production host. In this thesis a two-fold yield improvement, in overproduction of a membrane protein, Fpsl, is demonstrated, compared to two wild type strains.
This chapter summarizes the biochemistry and genetics of yeast carbohydrate metabolism. It mainly concentrates on data obtained for Saccharomyces cerevisiae and refers to other wine yeast only as far as important differences are concerned. The final part of this review describes the regulatory molecular mechanisms which govern glucose utilization.
A short reactivation period in aqueous media is required for active dry yeast (ADY) to be utilised in winemaking. Rehydration restores the active metabolic conditions necessary for good fermentative and competitive abilities. We used a reverse transcription-quantitative PCR (RT-qPCR) method with relative quantification to investigate the expression of seven hexose transporter genes (HXT1-7) and one invertase-encoding gene (SUC2) during ADY rehydration in water with or without sucrose. For this, seven candidate reference genes were evaluated, and the three most stably expressed genes were selected and used for mRNA normalisation. The results show that, during the rehydration in the presence of sucrose, yeast cells are able to immediately hydrolyse this sugar into glucose and fructose as soon as they are introduced in the medium. Subsequently, differential glucose/fructose uptake occurs, which is mediated by hexose transporters. At the transcriptomic level, there is a strong induction of the high-affinity transporters, HXT2 and HXT4, and the low-affinity transporters, HXT3 and HXT1, when ADY is rehydrated with sucrose, while HXT5 and HXT6/7 are expressed at high levels with a moderate tendency to decrease. In water, the HXT2 gene was the only one of the transporter genes studied that showed significant variations. These results suggest that during rehydration, expression is not simply regulated by the affinity to hexose but is also controlled by other mechanisms that allow the cell to bypass glucose control. Moreover, the expression of SUC2 showed little variation in media with sucrose, suggesting that other invertases and/or posttranscriptional controls exist.
Industrial yeast performance is often compromised during alcoholic fermentations due to bi-product inhibition. Ethanol is arguably the product with the greatest impact on
yeast performance, acting as a potent chemical stress on yeast cells. This stress eventually inhibits yeast growth and reduces ceil viability, therefore limiting alcohol
concentrations in the final product and increasing fermentation turnover times. The reduced cell growth rate and viability, as well as an increased growth lag period, are characteristic signs of cell stress. This is often accompanied at a molecular level by the induction of stress response genes. While there have been several investigations
into the effects of ethanol on yeast, few have focused on the underlying genetic mechanisms that enable yeast cells to tolerate and adapt to this stress. This thesis used
differential display and gene array technologies to determine, at a molecular genetics level, how yeast cells adapt to sub-lethal concentrations of ethanol. Such information is of fundamental importance to the development of yeast strains and strategies for the improvement of yeast performance in fermentation.
One of the major yeast stressors during fermentation is ethanol accumulation. Ethanol stress is associated with reduced cell growth and viability, consequently lowering yeast productivity. Although the underlying causes of ethanol inhibition of cells are yet to be identified, it has been discovered that yeast acclimatise more quickly to ethanol stress in the presence of low acetaldehyde concentrations; however, the biochemical processes underpinning this effect are unknown. The objective of this project was to identify the mechanisms associated with the acetaldehyde-mediated adaptation of yeast to ethanol stress, which may facilitate the development of yeast strains with improved ethanol tolerance and/or strategies for improving ethanol tolerance in yeast. Gene array analysis was used to study gene expression in Saccharomyces cerevisiae during acclimatisation to non-lethal ethanol stress, in the presence and absence of acetaldehyde. Acetaldehyde caused significant changes in gene expression in ethanol-stressed yeast. For example, many genes associated with protein biosynthesis were more highly expressed, as were pyruvate decarboxylase genes. Interestingly, however, there was no significant increase in the expression of trehalose synthesis genes or genes encoding HSPs; genes which, in previous studies, appeared to be associated with acclimatisation to ethanol-stress. In addition, acetaldehyde did not have a major impact on gene expression in non-stressed cultures. The results of this project are consistent with the speculation that the addition of acetaldehyde to ethanol-stressed S. cerevisiae primes glycolytic flux in ethanol-stressed cells by regenerating NAD+ from accumulated NADH. This, in turn, stimulates glyceraldehyde-3- phosphate dehydrogenase activity and might account for the acetaldehyde-mediated increased expression levels of pyruvate decarboxylase genes; elevated levels of pyruvate would potentially increase the need for PDC activity. Overall, these speculated effects of acetaldehyde on ethanol-stressed yeast would increase glycolytic rate and energy production.
The growth and glucose uptake of single cultures of the wine-related yeasts Kluyveromyces thermotolerans, Torulaspora delbrueckii, and Saccharomyces cerevisiae were investigated. The yeasts had different specific glucose uptake rates (q
s) that depended on the residual glucose concentration and the oxygen availability. In mixed cultures, the q
s values of the yeasts were not subject to any interaction effects over a wide range of glucose concentrations. Our results strongly indicate that the relative glucose uptake abilities of both non-Saccharomyces yeasts, i.e. the q
s(non-Saccharomyces)/q
s(S. cerevisiae) ratios, regulated their abilities to compete for space in mixed cultures with S. cerevisiae, which, in turn, regulated their early deaths. This hypothesis enabled us to explain why K. thermotolerans was less able than T. delbrueckii to coexist with S. cerevisiae in mixed cultures. Furthermore, it enabled us to explain why oxygen increased the abilities of K. thermotolerans and T. delbrueckii to coexist with S. cerevisiae in the mixed cultures.
During batch fermentation, the rate of ethanol production per milligram of cell protein is maximal for a brief period early in this process and declines progressively as ethanol accumulates in the surrounding broth. Our studies demonstrate that the removal of this accumulated ethanol does not immediately restore fermentative activity, and they provide evidence that the decline in metabolic rate is due to physiological changes (including possible ethanol damage) rather than to the presence of ethanol. Several potential causes for the decline in fermentative activity have been investigated. Viability remained at or above 90%, internal pH remained near neutrality, and the specific activities of the glycolytic and alcohologenic enzymes (measured in vitro) remained high throughout batch fermentation. None of these factors appears to be causally related to the fall in fermentative activity during batch fermentation.
A procedure for the rapid isolation of DNA from the yeast Saccharomyces cerevisiae is described. To release plasmid DNA for the transformation of Escherichia coli, cells are subjected to vortex mixing in the presence of acid-washed glass beads, Triton X-100, sodium dodecyl sulfate, phenol and chloroform. Centrifugation of this mixture separates the DNA from cellular debris. E. coli can be efficiently transformed with plasmid present in the aqueous layer without further purification of the plasmid DNA. This procedure also releases chromosomal DNA. Following two ethanol precipitations, the chromosomal DNA can be digested by restriction endonucleases and analysed by Southern blot analysis.
An approach to global restriction mapping is described that is applicable to any complex source DNA. By analyzing a single restriction digest for each member of a redundant set of lambda clones, a data base is constructed that contains fragment-size lists for all the clones. The clones are then grouped into subsets, each member of which is related to at least one other member by a significant overlap. Finally, a tree-searching algorithm seeks restriction maps that are consistent with the fragment-size lists for all the clones in each subset. The feasibility of the approach has been demonstrated by collecting data on 5000 lambda clones containing random 15-kilobase inserts of yeast DNA. It is shown that these data can be analyzed to produce regional maps of the yeast genome, extending in some cases for over 100 kilobases. In combination with hybridization probes to previously cloned genes, these local maps are already useful for defining the physical arrangement of closely linked genes. They may in the future serve as building blocks for the construction of a continuous global map.
The maltose transporter of Saccharomyces cerevisiae is rapidly degraded during fermentation in the absence of a nitrogen source. The location and mechanism of degradation of the transporter have been investigated. Using mutants defective in endocytosis, we have shown that degradation of this transporter requires internalization by endocytosis. In addition, studies of mutants defective in proteasome or vacuolar proteolysis revealed that degradation occurs in the vacuole and is independent of proteasome function. The results also revealed that degradation of the maltose transporter requires Sec18p and raised the question of whether in the absence of Sec18p activity the internalized maltose transporter is recycled back to the plasma membrane.
HXT4, a new member of the hexose transporter (HXT) family in Saccharomyces cerevisiae was identified by its ability to suppress the snf3 mutation in multicopy. Multicopy HXT4 increases both high and low affinity glucose transport in snf3 strains and increases low and high transport in wild-type strains. Characterization of HXT4 led to the discovery of a new class of multicopy suppressors of glucose transport defects: regulatory elements in the promoters of the HXT genes. We have designated these sequences DDSEs (DNA sequence dependent suppressing element). Multicopy HXT4 and DDSEs in the HXT1, HXT2, HXT3 and HXT4 promoters were found to restore growth to snf3 and grr1 strains on low glucose media. The DDSE in the HXT4 promoter was refined to a 340-bp sequence 450 bp upstream of the HXT4 translational start. This region was found to contain an 183-amino acid open reading frame. Extensive analysis indicates that the DNA sequence itself and not the encoded protein is responsible for suppression. The promoters of SNF3 and of other glycolytic genes examined did not suppress snf3 in multicopy. Suppression of snf3 by DDSE is dependent on the presence of either HXT2 or HXT3.
Expression of putative high-affinity glucose transport protein Hxt2p of Saccharomyces cerevisiae was repressed 15- to 20-fold in high concentrations of glucose or fructose. S. cerevisiae with either the ssn6-delta 9 or the hxk2-delta 1::URA3 mutation, each of which relieves glucose repression, exhibited high Hxt2p expression in both 2.0% glucose (normally repressing) and 0.05% glucose (normally derepressing) while S. cerevisiae with the snf1-delta 10 mutation, which causes constitutive repression, did not detectably express Hxt2p in either glucose concentration. In addition to repressing at high concentrations, glucose or fructose is required for induction of Hxt2p expression. Hxt2p was not expressed by wild-type S. cerevisiae in media containing only ethanol or galactose as carbon and energy source but was expressed if glucose was added. An hxk2-delta 1::URA3 mutant did not detectably express Hxt2p in ethanol or galactose, but an ssn6-delta9 mutant did highly express Hxt2p in both carbon sources. Thus, simple relief of glucose repression as occurs with hxk2 null mutants is insufficient for high-level Hxt2p expression. Mutation of ssn6, a general transcriptional repressor, does lead to Hxt2p expression in the absence of glucose induction, suggesting relief of an additional negative regulatory system. High expression of Hxt2p does not always result in HXT2-dependent high-affinity transport, implying that Hxt2p activity is regulated posttranslationally. In the high glucose condition for the ssn6 mutant, high-affinity glucose transport is derepressed. Deletion of the HXT2 locus does not diminish this level of transport. However, high-affinity glucose transport is diminished in the ssn6-delta9 hxt2 delta1 double mutant compared with ssn6-delta9 alone in low glucose. Thus, while constitutively expressed in ssn6 mutants, Hxt2p only appears to be active as a transporter under low-glucose conditions. Similarly, Hxt2p was found to be expressed under low-glucose conditions in an snf3 mutant which does not display high-affinity uptake. This finding suggests that SNF3 may be involved in the posttranslational regulation of Hxt2p.
Characterization and quantification of the Hxt2 (hexose transport) protein of Saccharomyces cerevisiae indicate that it is one of a set of differentially expressed high-affinity glucose transporters. The protein product of the HXT2 gene was specifically detected by antibodies raised against a synthetic peptide encompassing the 13 carboxyl-terminal amino acids predicted by the HXT2 gene sequence. Hxt2 migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a broad band or closely spaced doublet with an average M(r) of 47,000. Hxt2 cofractionated with the plasma membrane ATPase, Pma1, indicating that it is a plasma membrane protein. Hxt2 was not solubilized by high pH or urea but was solublized by detergents, which is characteristic of an integral membrane protein. Expression of the Hxt2 protein was measured under two different conditions that produce expression of high-affinity glucose transport: a medium shift from a high (2.0%) to a low (0.05%) glucose concentration (referred to below as high and low glucose) and growth from high to low glucose. Hxt2 as measured by immunoblotting increased 20-fold upon a shift from high-glucose to low-glucose medium, and the high-affinity glucose transport expressed had a strong HXT2-dependent component. Surprisingly, Hxt2 was not detectable when S. cerevisiae growing in high glucose approached glucose exhaustion, and the high-affinity glucose transport expressed under these conditions did not have an HXT2-dependent component. The role of Hxt2 in growth during aerobic batch culture in low-glucose medium was examined. An hxt2 null mutant grew and consumed glucose significantly more slowly than the wild type, and this phenotype correlated directly with appearance of the Hxt2 protein.
In Saccharomyces cerevisiae, TRK1 and TRK2 are required for high- and low-affinity K+ transport. Among suppressors of the K+ transport defect in trk1 delta trk2 delta cells, we have identified members of the sugar transporter gene superfamily. One suppressor encodes the previously identified glucose transporter HXT1, and another encodes a new member of this family, HXT3. The inferred amino acid sequence of HXT3 is 87% identical to that of HXT1, 64% identical to that of HXT2, and 32% identical to that of SNF3. Like HXT1 and HXT2, overexpression of HXT3 in snf3 delta cells confers growth on low-glucose or raffinose media. The function of another new member of the HXT superfamily, HXT4 (previously identified by its ability to suppress the snf3 delta phenotype; L. Bisson, personal communication), was revealed in experiments that deleted all possible combinations of the five members of the glucose transporter gene family. Neither SNF3, HXT1, HXT2, HXT3, nor HXT4 is essential for viability. snf3 delta hxt1 delta hxt2 delta hxt3 delta hxt4 delta cells are unable to grow on media containing high concentrations of glucose (5%) but can grow on low-glucose (0.5%) media, revealing the presence of a sixth transporter that is itself glucose repressible. This transporter may be negatively regulated by SNF3 since expression of SNF3 abolishes growth of hxt1 delta hxt2 delta hxt3 delta hxt4 delta cells on low-glucose medium. HXT1, HXT2, HXT3, and HXT4 can function independently: expression of any one of these genes is sufficient to confer growth on medium containing at least 1% glucose. A synergistic relationship between SNF3 and each of the HXT genes is suggested by the observation that SNF2 hxt1 delta hxt2 delta hxt3 delta hxt4 delta cells and snf3 delta HXT1 HXT2 HXT3 HXT4 cells are unable to grow on raffinose (low fructose) yet SNF3 in combination with any single HXT gene is sufficient for growth on raffinose. HXT1 and HXT3 are differentially regulated. HXT1::lacZ is maximally expressed during exponential growth whereas HXT3::lacZ is maximally expressed after entry into stationary phase.
The dominant kanr marker gene plays an important role in gene disruption experiments in budding yeast, as this marker can be used in a variety
of yeast strains lacking the conventional yeast markers. We have developed a loxP-kanMX-loxP gene disruption cassette, which combines the advantages of the heterologous kanr marker with those from the Cre-lox P recombination system. This disruption cassette integrates with high efficiency via homologous integration at the correct
genomic locus (routinely 70%). Upon expression of the Cre recombinase the kanMX module is excised by an efficient recombination between the loxP sites, leaving behind a single loxP site at the chromosomal locus. This system allows repeated use of the kanr marker gene and will be of great advantage for the functional analysis of gene families.
When Saccharomyces cerevisiae cells growing on galactose are transferred onto glucose medium containing cycloheximide, an inhibitor of protein synthesis, a rapid reduction of Gal2p-mediated galactose uptake is observed. We show that glucose-induced inactivation of Gal2p is due to its degradation. Stabilization of Gal2p in pra1 mutant cells devoid of vacuolar proteinase activity is observed. Subcellular fractionation and indirect immunofluorescence showed that the Gal2 transporter accumulates in the vacuole of the mutant cells, directly demonstrating that its degradation requires vacuolar proteolysis. In contrast, Gal2p degradation is proteasome independent since its half-life is unaffected in pre1-1 pre2-2, cim3-1, and cim5-1 mutants defective in several subunits of the protease complex. In addition, vacuolar delivery of Gal2p was shown to be blocked in conditional end3 and end4 mutants at the nonpermissive temperature, indicating that delivery of Gal2p to the vacuole occurs via the endocytic pathway. Taken together, the results presented here demonstrate that glucose-induced proteolysis of Gal2p is dependent on endocytosis and vacuolar proteolysis and is independent of the functional proteasome. Moreover, we show that Gal2p is ubiquitinated under conditions of glucose-induced inactivation.
The kinetics of glucose transport and the transcription of all 20 members of the HXT hexose transporter gene family were studied in relation to the steady state in situ carbon metabolism of Saccharomyces cerevisiae CEN.PK113-7D grown in chemostat cultures. Cells were cultivated at a dilution rate of 0.10 h-1 under various nutrient-limited conditions (anaerobically glucose- or nitrogen-limited or aerobically glucose-, galactose-, fructose-, ethanol-, or nitrogen-limited), or at dilution rates ranging between 0.05 and 0.38 h-1 in aerobic glucose-limited cultures. Transcription of HXT1-HXT7 was correlated with the extracellular glucose concentration in the cultures. Transcription of GAL2, encoding the galactose transporter, was only detected in galactose-limited cultures. SNF3 and RGT2, two members of the HXT family that encode glucose sensors, were transcribed at low levels. HXT8-HXT17 transcripts were detected at very low levels. A consistent relationship was observed between the expression of individual HXT genes and the glucose transport kinetics determined from zero-trans influx of 14C-glucose during 5 s. This relationship was in broad agreement with the transport kinetics of Hxt1-Hxt7 and Gal2 deduced in previous studies on single-HXT strains. At lower dilution rates the glucose transport capacity estimated from zero-trans influx experiments and the residual glucose concentration exceeded the measured in situ glucose consumption rate. At high dilution rates, however, the estimated glucose transport capacity was too low to account for the in situ glucose consumption rate.
We have analyzed the transcriptional response to osmotic shock in the yeast Saccharomyces cerevisiae. The mRNA level of 186 genes increased at least 3-fold after a shift to NaCl or sorbitol, whereas that of more than 100 genes was at least 1.5-fold diminished. Many induced genes encode proteins that presumably contribute to protection against different types of damage or encode enzymes in glycerol, trehalose, and glycogen metabolism. Several genes, which encode poorly expressed isoforms of enzymes in carbohydrate metabolism, were induced. The high osmolarity glycerol (HOG) pathway is required for full induction of many but not all genes. The recently characterized Hot1p transcription factor is required for normal expression of a subset of the HOG pathway-dependent responses. Stimulated expression of the genes that required the general stress-response transcription factors Msn2p and Msn4p was also reduced in a hog1 mutant, suggesting that Msn2p/Msn4p might be regulated by the HOG pathway. The expression of genes that are known to be controlled by the mating pheromone response pathway was stimulated by osmotic shock specifically in a hog1 mutant. Inappropriate activation of the mating response may contribute to the growth defect of a hog1 mutant in high osmolarity medium.
Glucose, the most abundant monosaccharide in nature, is the principal carbon and energy source for nearly all cells. The first, and rate-limiting, step of glucose metabolism is its transport across the plasma membrane. In cells of many organisms glucose ensures its own efficient metabolism by serving as an environmental stimulus that regulates the quantity, types, and activity of glucose transporters, both at the transcriptional and posttranslational levels. This is most apparent in the baker’s yeast Saccharomyces cerevisiae, which has 20 genes encoding known or likely glucose transporters, each of which is known or likely to have a different affinity for glucose. The expression and function of most of these HXT genes is regulated by different levels of glucose. This review focuses on the mechanisms S. cerevisiae and a few other fungal species utilize for sensing the level of glucose and transmitting this information to the nucleus to alter HXT gene expression. One mechanism represses transcription of some HXT genes when glucose levels are high and works through the Mig1 transcriptional repressor, whose function is regulated by the Snf1-Snf4 protein kinase and Reg1-Glc7 protein phosphatase. Another pathway induces HXT expression in response to glucose and employs the Rgt1 transcriptional repressor, a ubiquitin ligase protein complex (SCF Grr1 ) that regulates Rgt1 function, and two glucose sensors in the membrane (Snf3 and Rgt2) that bind glucose and generate the intracellular signal to which Rgt1 responds. These two regulatory pathways collaborate with other, less well-understood, pathways to ensure that yeast cells express the glucose transporters best suited for the amount of glucose available.
This chapter reviews the effects of alcohols on microorganisms. Alcohols are ubiquitous small molecules, which are produced both chemically and as products of microbial fermentation. Accumulation of alcohols in the microbial environment represents a form of environmental stress, analogous to extremes in pH value and temperature. The chapter discusses the action of ethanol and other alcohols on microorganisms, explains several important mechanisms of action. Alcohols have been employed for many years both as a disinfectant and as a preservative. Concentrations of ethanol above 15% result in immediate inactivation of most vegetative organisms, with spores being considerably more resistant. Low concentrations of ethanol also render bacteria more sensitive to inactivation by ionizing radiation and by lipophilic acids. The chapter concludes that the basic actions of alcohols on both eukaryotic and prokaryotic organisms share the same general principles. These effects appear to be dominated by the physicochemical properties of alcohols rather than involving specific receptors. All hydrophobic and electrostatic interactions in the cytosolic and envelope components of cells can potentially be affected. These include membranes, conformations of enzymes and macromolecules, activity coefficients of metabolites, permitivity, ionization potentials, pK values of functional groups, and pH value.
In Saccharomyces cerevisiae, hexose uptake is mediated by HXT proteins which belong to a superfamily of monosaccharide facilitators. We have identified three more genes that encode hexose transporters (HXT5, 6, 7). Genes HXT6 and HXT7 are almost identical and located in tandem 3′ adjacent to HXT3 on chromosome IV. We have constructed a set of congenic strains expressing none or any one of the seven known HXT genes and followed growth and flux rates for glucose utilization. The hxt null strain does not grow on glucose, fructose or mannose, and both glucose uptake and flux rate were below the detection level. Expression of either HXT1, 2, 3, 4, 6 or 7 is basically sufficient for aerobic growth on these sugars. In most of the constructs, glucose was the preferred substrate compared to fructose or mannose. There is a considerable variation in flux and growth rates with 1% glucose, dependent on the expression of the individual HXT genes. Expression of either HXT2, 6 or 7 in the null background is sufficient for growth on 0.1% glucose, while growth of strains with only HXT1, 3 or 4 requires higher (≥1%) glucose concentrations. These results demonstrate that individual HXT proteins can function independently as hexose transporters, and that most of the metabolically relevant HXT transporters from S. cerevisiae have been identified.
Premature arrest of fermentation constitutes one of the most challenging problems in wine production. The causes of stuck and sluggish fermentations are numerous, troublesome to diagnose, and difficult to rectify. It has become well-established that fermentation rate decreases due to a targeted loss of hexose transport capacity. Factors which have been correlated with incomplete fermentations also regulate transporter expression, turnover and function. Several causes of slow and incomplete fermentations, including ethanol toxicity, have been well-characterized under enological conditions as described herein. Other potential factors that may impact cell growth, viability, and fermentative activity have not been sufficiently evaluated. The role of these factors in problematic enological fermentations is discussed.
Ethanol, isopropanol, propanol and butanol exponentially inhibited the maximum velocity of the glucose transport system ofSaccharomyces cerevisiae, determined by use of the non-metabolizable analogued-xylose. While the exponential inhibition constants increased with the lipid solubility of the alkanols, they were independent of temperature in the range 21–35C: the Arrhenius plots (modified according to the theory of absolute reaction rates) of the initial maximum rates of xylose transport were linear and parallel in both the absence and presence of alkanols. Thus, the alkanols did not affect the enthalpy of activation of the glucose transport system (H
was 12 190 cal mol-1) but decreased the entropy of activation. The following entropy coefficients (decrease in activation entropy per unit concentration of alkanol) were obtained: ethanol,-0.84; isopropanol,-1.21; propanol,-1.41 and butanol,-3.18 entropy units per mole per liter. The temperature relations of glucose fermentation with and without ethanol by resting cells over the temperature range studied (15–35C) were nearly identical with those of the glucose transport system, suggesting that the latter mediates the rate-limiting step of the former and that this relationship is maintained in the presence of ethanol.
To better describe the importance of initial assimilable nitrogen content on the kinetics of alcoholic fermentation and the effectiveness of ammonium additions, a study was done using an automatic device for fermentation monitoring. A good correlation was found between the maximum CO2 production rate, obtained early during the fermentation, and the assimilable nitrogen content of the must. So it is possible to use this kinetic parameter to detect nitrogen deficiencies. For industrial applications, a threshold value of 1.3 gCO2/l·h at 24°C (corresponding to about 140 mg N/l) is proposed. In the case of deficient must, a nitrogen addition during the fermentation rapidly increased the CO2 production rate and reduced the fermentation duration. This reduction, which may be approximately predicted from (dCO2/dt)max, is the same, provided that the addition is made before the halfway point of the fermentation.
Populations of cells suspended anaerobically in buffered (pH 4.5) M ethanol remained viable to a greater extent when their plasma membranes were enriched in linoleyl rather than oleyl residues irrespective of the nature of the sterol enrichment. However, populations with membranes enriched in ergosterol or stigmasterol and linoleyl residues were more resistant to ethanol than populations enriched in campesterol or cholesterol and linoleyl residues. Populations enriched in ergosterol and cetoleic acid lost viability at about the same rate as those enriched in oleyl residues, while populations grown in the presence of this sterol and palmitoleic acid were more resistant to ethanol. Suspending cells in buffered ethanol for up to 24 h did not lower the ethanol concentration.
Incorporation of ethanol (1.0 or 1.25 M) into exponential-phase cultures of Saccharomyces cerevisiae NCYC 366 growing anaerobically in a medium supplemented with ergosterol and an unsaturated fatty acid caused a retardation in growth rat, which was greater when the medium contained oleic rather than linoleic acid. Ethanol incorporation led to an immediate drop in growth rate, and ethanol-containing cultures grew at the slower rate for at least 10 h. Incorporation of ethanol (0.5 M) into buffered (pH 4.5) cell suspensions containing D-[6-3H] glucose, D-[1-14C] glucosamine, L-[U-14C] lysine or arginine, or KH232PO4 lowered the rate of solute accumulation by cells. Rates of accumulation of glucose, lysine and arginine were retarded to a greater extent when cells had been grown in the presence of oleic rather than linoleic acid. This difference was not observed with accumulation of phosphate. Ethanol was extracted from exponential-phase cells by four different methods. Cells grown in the presence of linoleic acid contained a slightly, but consistently, lower concentration of ethanol than cells grown in oleic acid-containing medium. The ethanol concentration in cells was 5-7 times greater than that in the cell-free medium.
The stability of the K+ transport system in Saccharomyces cerevisiae has been studied upon inhibition of protein synthesis with cycloheximide. Addition of the antibiotic gave rise to an inactivation of this transport. This activation followed first-order kinetics and was stimulated by the presence of a fermentable substrate. A half-life of about 4 h could be calculated in the presence of glucose. The results indicate that, similarly to sugar carriers, K+ transport system is less stable than the bulk of proteins of this organism.
A series of yeast shuttle vectors and host strains has been created to allow more efficient manipulation of DNA in Saccharomyces cerevisiae. Transplacement vectors were constructed and used to derive yeast strains containing nonreverting his3, trp1, leu2 and ura3 mutations. A set of YCp and YIp vectors (pRS series) was then made based on the backbone of the multipurpose plasmid pBLUESCRIPT. These pRS vectors are all uniform in structure and differ only in the yeast selectable marker gene used (HIS3, TRP1, LEU2 and URA3). They possess all of the attributes of pBLUESCRIPT and several yeast-specific features as well. Using a pRS vector, one can perform most standard DNA manipulations in the same plasmid that is introduced into yeast.
The sugar transport systems of Saccharomyces cerevisiae are irreversibly inactivated when protein synthesis is inhibited. This inactivation is responsible for the drastic decrease in fermentation observed in ammonium-starved yeast and is related to the occurrence of the Pasteur effect in these cells. Our study of the inactivation of the glucose transport system indicates that both the high-affinity and the low-affinity components of this system are inactivated. Inactivation of the high-affinity component evidently requires the utilization of a fermentable substrate by the cells, since inactivation did not occur during carbon starvation, when a fermentable sugar was added to starved cells, inactivation began, when the fermentation inhibitors iodoacetate or arsenate were added in addition to sugars, the inactivation was prevented, when a non-fermentable substrate was added instead of sugars, inactivation was also prevented. The inactivation of the low-affinity component appeared to show similar requirements. It is concluded that the glucose transport system in S. cerevisiae is regulated by a catabolite-inactivation process.
1.
Saturation curves for the initial rates of uptake of non-fermentable sugars and for fermentable ones in normal and iodoacetate-treated cells have been obtained with baker's yeast harvested in log phase. The Km values for each of the sugars tested were found to be 2 to 10 times lower in the presence of fermentation than in its absence. The same effect has been observed in efflux measurements.
2.
The kinetic properties of transport under conditions in which the latter is largely inactivated by uranyl ions are similar to those in untreated cells in the case of glucose and fructose and markedly different in the case of mannose.
3.
The saturation curves appear biphasic in double reciprocal plots under certain conditions (for instance, cells treated with iodoacetic acid and uranyl ions, or cells washed a few seconds after their contact with sugars). Under these conditions two Km values have been calculated for glucose, mannose and xylose.
4.
In steady fermentation of mannose, whose transport seems to be in large potential excess over phosphorylation, the intracellular concentration of free sugar is nevertheless much lower than that corresponding to equilibration by an excess of transport over phosphorylation. In cells in which hexokinase activity is depleted by treatment with xylose, mannose accumulates to near equilibrium levels with the outside sugar.
5.
The kinetics of aerobic fermentation of glucose in respiring cells show an apparent Km one order of magnitude higher than that corresponding to anaerobic fermentation.
6.
The above observations are interpreted as indicating that the constitutive transport system for sugars can exist in two states showing different affinities for sugars. In the absence of fermentation or in aerobic fermentation in respiring cells the state of higher Km prevails, while in the presence of anaerobic fermentation the state of lower Km prevails. Under certain conditions the two states can coexist to the point of giving rise to biphasic saturation curves. The evidence for two affinity states in the carrier and the fact that mannose is fermented at the same maximal rate as glucose or fructose, in spite of the marked differences in their kinetic parameters of transport and phosphorylation, are interpreted as consistent with the hypothesis of the existence of a regulatory feed-back mechanism responsive to the level of an intermediary metabolite of glycolysis.
Yeast cells can respond and adapt to osmotic stress. In our attempt to clarify the molecular mechanisms of cellular responses to osmotic stress, we cloned seven cDNAs for hyperosmolarity-responsive (HOR) genes from Saccharomyces cerevisiae by a differential screening method. Structural analysis of the clones revealed that those designated HOR1, HOR3, HOR4, HOR5 and HOR6 encoded glycerol-3-phosphate dehydrogenase (Gpd1p), glucokinase (Glk1p), hexose transporter (Hxt1p), heat-shock protein 12 (Hsp12p) and Na+, K+, Li(+)-ATPase (Ena1p), respectively. HOR2 and HOR7 corresponded to novel genes. Gpd1p is a key enzyme in the synthesis of glycerol, which is a major osmoprotectant in S. cerevisiae. Cloning of HOR1/GPD1 as a HOR gene indicates that the accumulation of glycerol in yeast cells under hyperosmotic stress is, at least in part, caused by an increase in the level of GPDH protein. We performed a series of Northern blot analyses using HOR cDNAs as probes and RNAs prepared from cells grown under various conditions and from various mutant cells. The results suggested that all the HOR genes are regulated by common signal transduction pathways. However, the fact that they exhibited certain distinct responses indicated that they might also be regulated by specific pathways in addition to the common pathways. Ca2+ seemed to be involved in the signaling systems. In addition, Hog1p, one of the MAP kinases in yeast, appeared to be involved in the regulation of expression of HOR genes, although its function seemed to be insufficient for the overall regulation of expression of these genes.
In Saccharomyces cerevisiae, hexose uptake is mediated by HXT proteins which belong to a superfamily of monosaccharide facilitators. We have identified three more genes that encode hexose transporters (HXT5, 6, 7). Genes HXT6 and HXT7 are almost identical and located in tandem 3' adjacent to HXT3 on chromosome IV. We have constructed a set of congenic strains expressing none or any one of the seven known HXT genes and followed growth and flux rates for glucose utilization. The hxt null strain does not grow on glucose, fructose or mannose, and both glucose uptake and flux rate were below the detection level. Expression of either HXT1, 2, 3, 4, 6 or 7 is basically sufficient for aerobic growth on these sugars. In most of the constructs, glucose was the preferred substrate compared to fructose or mannose. There is a considerable variation in flux and growth rates with 1% glucose, dependent on the expression of the individual HXT genes. Expression of either HXT2, 6 or 7 in the null background is sufficient for growth on 0.1% glucose, while growth of strains with only HXT1, 3 or 4 requires higher (> or = 1%) glucose concentrations. These results demonstrate that individual HXT proteins can function independently as hexose transporters, and that most of the metabolically relevant HXT transporters from S. cerevisiae have been identified.
The HXT genes (HXT1 to HXT4) of the yeast Saccharomyces cerevisiae encode hexose transporters. We found that transcription of these genes is induced 10- to 300-fold by glucose. Analysis of glucose induction of HXT gene expression revealed three types of regulation: (i) induction by glucose independent of sugar concentration (HXT3); (ii) induction by low levels of glucose and repression at high glucose concentrations (HXT2 and HXT4); and (iii) induction only at high glucose concentrations (HXT1). The lack of expression of all four HXT genes in the absence of glucose is due to a repression mechanism that requires Rgt1p and Ssn6p. GRR1 seems to encode a positive regulator of HXT expression, since grr1 mutants are defective in glucose induction of all four HXT genes. Mutations in RGT1 suppress the defect in HXT expression caused by grr1 mutations, leading us to propose that glucose induces HXT expression by activating Grr1p, which inhibits the function of the Rgt1p repressor. HXT1 expression is also induced by high glucose levels through another regulatory mechanism: rgt1 mutants still require high levels of glucose for maximal induction of HXT1 expression. The lack of induction of HXT2 and HXT4 expression on high levels of glucose is due to glucose repression: these genes become induced at high glucose concentrations in glucose repression mutants (hxk2, reg1, ssn6, tup1, or mig1). Components of the glucose repression pathway (Hxk2p and Reg1p) are also required for generation of the high-level glucose induction signal for expression of the HXT1 gene. Thus, the glucose repression and glucose induction mechanisms share some of the same components and may share the same primary signal generated from glucose.
The possible relationship between endocytosis and catabolite inactivation of plasma membrane proteins in Saccharomyces cerevisiae has been investigated. Using mutants with an increased rate of endocytosis we have shown that there is a positive correlation between the rate of endocytosis and the rate of inactivation of the K+ and glucose transport systems. It is concluded that endocytosis is involved in catabolite inactivation of these two transport systems.
A low-affinity glucose transporter gene of Saccharomyces cerevisiae was cloned by complementation of the rag1 mutation in a strain of Kluyveromyces lactis defective in low-affinity glucose transport. Gene sequence and effects of null mutation in S. cerevisiae were described. Data indicated that there are multiple genes for low-affinity glucose transport.
The yeast Saccharomyces cerevisiae consumes mono- and disaccharides preferentially to any other carbon source. Since sugars do not freely permeate biological membranes, cellular uptake of these compounds requires the action of 'transporters'. The purpose of this review is to summarize the present knowledge on sugar transport in this organism. Yeast cells show two transporters for monosaccharides, the so-called glucose and galactose transporters that act by a facilitated diffusion mechanism. In the case of glucose transport, which also acts upon D-fructose and D-mannose, two components with high- and low-affinity constants have been identified kinetically. Activity of the high-affinity component is dependent on the presence of active kinases whereas activity of the low-affinity component is independent of the presence of these enzymes. Three genes, SNF3, HXT1 and HXT2, encode three different glucose transporters with a high affinity for the substrates and are repressed by high concentrations of glucose in the medium. Kinetic studies suggest that at least one additional gene exists that encodes a transporter with a low affinity and is expressed constitutively. The present view is that there are several additional transporters for glucose that have not yet been identified. Galactose transport has only one natural substrate, D-galactose, and is encoded by the gene GAL2. Expression of this gene is induced by galactose and repressed by glucose. Two transporters for disaccharides have been identified in S. cerevisiae: maltose and alpha-methylglucoside transporters. These transporters are H(+)-symports that depend on the electrochemical proton gradient and are independent of the ATP level. The gene that encodes the maltose transporter is clustered with the other two genes required for maltose utilization in a locus that is found repeated at different chromosomal locations. Its expression is induced by maltose and repressed by glucose. The rate of sugar uptake in yeast cells is controlled by changes in affinity of the corresponding transporters as well as by an irreversible inactivation that affects their Vmax. The mechanisms involved in these regulatory processes are unknown at present.
Transport of sugars is a fundamental property of all eukaryotic cells. Of particular importance is the uptake of glucose, a preferred carbon and energy source. The rate of glucose utilization in yeast is often dictated by the activity and concentration of glucose transporters in the plasma membrane. Given the importance of transport as a site of control of glycolytic flux, the regulation of glucose transporters is necessarily complex. The molecular analysis of these transporters in Saccharomyces has revealed the existence of a multigene family of sugar carriers. Recent data have raised the question of the actual role of all of these proteins in sugar catabolism, as some appear to be lowly expressed, and point mutations of these genes may confer pleiotropic phenotypes, inconsistent with a simple role as catabolic transporters. The transporters themselves appear to be intimately involved in the process of sensing glucose, a model for which there is growing support.
Transcription of the yeast HXT2 and HXT4 genes, which encode glucose transporters, is induced only by low levels of glucose.
This low-glucose-induced expression is mediated by two independent repression mechanisms: in the absence of glucose, transcription
of both genes is prevented by Rgt1p, a C6 zinc cluster protein; at high levels of glucose, expression of HXT2 and HXT4 is
repressed by Mig1p. Only at low glucose concentrations are both repressors inactive, leading to a 10- to 20-fold induction
of gene expression. Mig1p and Rgt1p act directly on HXT2 and HXT4 by binding to their promoters. This transcriptional regulation
is physiologically very important to the yeast cell because it causes these glucose transporters to be expressed only in low-glucose
media, in which they are required for growth.
Saccharomyces cerevisiae accomplishes high rates of hexose transport. The kinetics of hexose transport are complex. The capacity and kinetic complexity of hexose transport in yeast are reflected in the large number of sugar transporter genes in the genome. Twenty hexose transporter genes exist in S. cerevisiae. Some of these have been found by genetic means; many have been discovered by the comprehensive sequencing of the yeast genome. This review codifies the nomenclature of the hexose transporter genes and describes the sequence homology and structural similarity of the proteins they encode. Information about the expression and function of the transporters is presented. Access to the sequences of the genes and proteins at three sequence databases is provided via the World Wide Web.
In Saccharomyces cerevisiae, there are a large number of genes (HXT1-HXT17/SNF3/RGT2) encoding putative hexose transporters which, together with a galactose permease gene (GAL2), belong to a superfamily of monosaccharide facilitator genes. We have performed a systematic analysis of the HXT1-7 and GAL2 genes and their function in hexose transport. Glucose uptake was below the detection level in the hxt1-7 null strain growing on maltose. Determination of the kinetic parameters of individual hexose transporter-related proteins (Hxtp) expressed in the hxt null background revealed Hxt1p and Hxt3p as low-affinity transporters (Km(glucose) = 50-100 mM), Hxt2p and Hxt4p as moderately low in affinity (Km(glucose) about 10 mM), and Hxt6p, Hxt7p as well as Gal2p as high-affinity transporters (Km(glucosse) = 1-2 mM). However, Hxt2p kinetics in cells grown on low glucose concentrations showed a high-affinity (Km = 1.5 mM) and a low-affinity component (Km = 60 mM). Furthermore, we investigated the involvement of glucose transport in glucose signalling. Glucose repression of MAL2, SUC2 and GAL1 was not dependent on a specific transporter but, instead, the strength of the repression signal was dependent on the level of expression, the properties of the individual transporters and the kind of sugar transported. The strength of the glucose repression signal correlated with the glucose consumption rates in the different strains, indicating that glucose transport limits the provision of a triggering signal rather then being directly involved in the triggering mechanism.
Transport across the plasma membrane is the first, obligatory step of hexose utilization. In yeast cells the uptake of hexoses is mediated by a large family of related transporter proteins. In baker's yeast Saccharomyces cerevisiae the genes of 20 different hexose transporter-related proteins have been identified. Six of these transmembrane proteins mediate the metabolically relevant uptake of glucose, fructose and mannose for growth, two others catalyze the transport of only small amounts of these sugars, one protein is a galactose transporter but also able to transport glucose, two transporters act as glucose sensors, two others are involved in the pleiotropic drug resistance process, and the functions of the remaining hexose transporter-related proteins are not yet known. The catabolic hexose transporters exhibit different affinities for their substrates, and expression of their corresponding genes is controlled by the glucose sensors according to the availability of carbon sources. In contrast, milk yeast Kluyveromyces lactis contains only a few different hexose transporters. Genes of other monosaccharide transporter-related proteins have been found in fission yeast Schizosaccharomyces pombe and in the xylose-fermenting yeast Pichia stipitis. However, the molecular genetics of hexose transport in many other yeasts remains to be established. The further characterization of this multigene family of hexose transporters should help to elucidate the role of transport in yeast sugar metabolism.
Addition of glucose to Saccharomyces cerevisiae inactivates the maltose transporter. The general consensus is that this inactivation, called catabolite inactivation, is one of the control mechanisms developed by this organism to use glucose preferentially whenever it is available. Using nitrogen-starved cells (resting cells), it has been shown that glucose triggers endocytosis and degradation of the transporter in the vacuole. We now show that maltose itself triggers inactivation and degradation of its own transporter as efficiently as glucose. This fact, and the observation that glucose inactivates a variety of plasma membrane proteins including glucose transporters themselves, suggests that catabolite inactivation of the maltose transporter in nitrogen-starved cells is not a control mechanism specifically directed to ensure a preferential use of glucose. It is proposed that, in this metabolic condition, inactivation of the maltose transporter might be due to the stimulation of the general protein turnover that follows nitrogen starvation.
After addition of high concentrations of glucose, rates of high-affinity glucose uptake in Saccharomyces cerevisiae decrease rapidly. We found that the high-affinity hexose transporters Hxt6 and Hxt7 are subject to glucose-induced proteolytic degradation (catabolite inactivation). Degradation occurs in the vacuole, as Hxt6/7 were stabilized in proteinase A-deficient mutant cells. Degradation was independent of the proteasome. The half-life of Hxt6 and Hxt7 strongly increased in end4, ren1 and act1 mutant strains, indicating that the proteins are delivered to the vacuole by endocytosis. Moreover, both proteins were also stabilized in mutants defective in ubiquitination. However, the initial signal that triggers catabolite inactivation is not relayed via the glucose sensors Snf3 and Rgt2.
Glucose, the most abundant monosaccharide in nature, is the principal carbon and energy source for nearly all cells. The first, and rate-limiting, step of glucose metabolism is its transport across the plasma membrane. In cells of many organisms glucose ensures its own efficient metabolism by serving as an environmental stimulus that regulates the quantity, types, and activity of glucose transporters, both at the transcriptional and posttranslational levels. This is most apparent in the baker's yeast Saccharomyces cerevisiae, which has 20 genes encoding known or likely glucose transporters, each of which is known or likely to have a different affinity for glucose. The expression and function of most of these HXT genes is regulated by different levels of glucose. This review focuses on the mechanisms S. cerevisiae and a few other fungal species utilize for sensing the level of glucose and transmitting this information to the nucleus to alter HXT gene expression. One mechanism represses transcription of some HXT genes when glucose levels are high and works through the Mig1 transcriptional repressor, whose function is regulated by the Snf1-Snf4 protein kinase and Reg1-Glc7 protein phosphatase. Another pathway induces HXT expression in response to glucose and employs the Rgt1 transcriptional repressor, a ubiquitin ligase protein complex (SCF(Grr1)) that regulates Rgt1 function, and two glucose sensors in the membrane (Snf3 and Rgt2) that bind glucose and generate the intracellular signal to which Rgt1 responds. These two regulatory pathways collaborate with other, less well-understood, pathways to ensure that yeast cells express the glucose transporters best suited for the amount of glucose available.
The hexose transporter family of Saccharomyces cerevisiae comprises 18 proteins (Hxt1-17, Gal2). Here, we demonstrate that all these proteins, except Hxt12, and additionally three members of the maltose transporter family (Agt1, Ydl247, Yjr160) are able to transport hexoses. In a yeast strain deleted for HXT1-17, GAL2, AGT1, YDL247w and YJR160c, glucose consumption and transport activity were completely abolished. However, as additional deletion of the glucose sensor gene SNF3 partially restored growth on hexoses, our data indicate the existence of even more proteins able to transport hexoses in yeast.