Α-glucoside Transporters Research Articles

Specialization restricts the evolutionary paths available to yeast sugar transporters.

Functional innovation at the protein level is a key source of evolutionary novelties. The constraints on functional innovations are likely to be highly specific in different proteins, which are shaped by their unique histories and the extent of global epistasis that arises from their structures and biochemistries. These contextual nuances in the sequence-function relationship have implications both for a basic understanding of the evolutionary process and for engineering proteins with desirable properties. Here, we have investigated the molecular basis of novel function in a model member of an ancient, conserved, and biotechnologically relevant protein family. These Major Facilitator Superfamily sugar porters are a functionally diverse group of proteins that are thought to be highly plastic and evolvable. By dissecting a recent evolutionary innovation in an α-glucoside transporter from the yeast Saccharomyces eubayanus, we show that the ability to transport a novel substrate requires high-order interactions between many protein regions and numerous specific residues proximal to the transport channel. To reconcile the functional diversity of this family with the constrained evolution of this model protein, we generated new, state-of-the-art genome annotations for 332 Saccharomycotina yeast species spanning approximately 400 million years of evolution. By integrating phylogenetic and phenotypic analyses across these species, we show that the model yeast α-glucoside transporters likely evolved from a multifunctional ancestor and became subfunctionalized. The accumulation of additive and epistatic substitutions likely entrenched this subfunction, which made the simultaneous acquisition of multiple interacting substitutions the only reasonably accessible path to novelty.

Contrasting Strategies for Sucrose Utilization in a Floral Yeast Clade.

ABSTRACTYeast species in the Wickerhamiella and Starmerella genera (W/S clade) thrive in the sugar-rich floral niche. We have previously shown that species belonging to this clade harbor an unparalleled number of genes of bacterial origin, among which is the SUC2 gene, encoding a sucrose-hydrolyzing enzyme. In this study, we used complementary in silico and experimental approaches to examine sucrose utilization in a broader cohort of species representing extant diversity in the W/S clade. Distinct strategies and modes of sucrose assimilation were unveiled, involving either extracellular sucrose hydrolysis through secreted bacterial Suc2 or intracellular assimilation using broad-substrate-range α-glucoside/H+ symporters and α-glucosidases. The intracellular pathway is encoded in two types of gene clusters reminiscent of the MAL clusters in Saccharomyces cerevisiae, where they are involved in maltose utilization. The genes composing each of the two types of MAL clusters found in the W/S clade have disparate evolutionary histories, suggesting that they formed de novo. Both transporters and glucosidases were shown to be functional and additionally involved in the metabolization of other disaccharides, such as maltose and melezitose. In one Wickerhamiella species lacking the α-glucoside transporter, maltose assimilation is accomplished extracellularly, an attribute which has been rarely observed in fungi. Sucrose assimilation in Wickerhamiella generally escaped both glucose repression and the need for an activator and is thus essentially constitutive, which is consistent with the abundance of both glucose and sucrose in the floral niche. The notable plasticity associated with disaccharide utilization in the W/S clade is discussed in the context of ecological implications and energy metabolism.IMPORTANCE Microbes usually have flexible metabolic capabilities and are able to use different compounds to meet their needs. The yeasts belonging to the Wickerhamiella and Starmerella genera (forming the so-called W/S clade) are usually found in flowers or insects that visit flowers and are known for having acquired many genes from bacteria by a process called horizontal gene transfer. One such gene, dubbed SUC2, is used to assimilate sucrose, which is one of the most abundant sugars in floral nectar. Here, we show that different lineages within the W/S clade used different solutions for sucrose utilization that dispensed SUC2 and differed in their energy requirements, in their capacity to scavenge small amounts of sucrose from the environment, and in the potential for sharing this resource with other microbial species. We posit that this plasticity is possibly dictated by adaptation to the specific requirements of each species.

Improving the Utilization of Isomaltose and Panose by Lager Yeast Saccharomyces pastorianus

Approximately 25% of all carbohydrates in industrial worts are poorly, if at all, fermented by brewing yeast. This includes dextrins, β-glucans, arabinose, xylose, disaccharides such as isomaltose, nigerose, kojibiose, and trisaccharides such as panose and isopanose. As the efficient utilization of carbohydrates during the wort’s fermentation impacts the alcohol yield and the organoleptic traits of the product, developing brewing strains with enhanced abilities to ferment subsets of these sugars is highly desirable. In this study, we developed Saccharomyces pastorianus laboratory yeast strains with a superior capacity to grow on isomaltose and panose. First, we designed a plasmid toolbox for the stable integration of genes into lager strains. Next, we used the toolbox to elevate the levels of the α-glucoside transporter Agt1 and the major isomaltase Ima1. This was achieved by integrating synthetic AGT1 and IMA1 genes under the control of strong constitutive promoters into defined genomic sites. As a result, strains carrying both genes showed a superior capacity to grow on panose and isomaltose, indicating that Ima1 and Agt1 act in synergy to consume these sugars. Our study suggests that non-GMO strategies aiming to develop strains with improved isomaltose and panose utilization could include identifying strains that overexpress AGT1 and IMA1.

Genome Mining of Non-Conventional Yeasts: Search and Analysis of MAL Clusters and Proteins.

Genomic clustering of functionally related genes is rare in yeasts and other eukaryotes with only few examples available. Here, we summarize our data on a nontelomeric MAL cluster of a non-conventional methylotrophic yeast Ogataea (Hansenula) polymorpha containing genes for α-glucosidase MAL1, α-glucoside permease MAL2 and two hypothetical transcriptional activators. Using genome mining, we detected MAL clusters of varied number, position and composition in many other maltose-assimilating non-conventional yeasts from different phylogenetic groups. The highest number of MAL clusters was detected in Lipomyces starkeyi while no MAL clusters were found in Schizosaccharomyces pombe and Blastobotrys adeninivorans. Phylograms of α-glucosidases and α-glucoside transporters of yeasts agreed with phylogenesis of the respective yeast species. Substrate specificity of unstudied α-glucosidases was predicted from protein sequence analysis. Specific activities of Scheffersomyces stipitis α-glucosidases MAL7, MAL8, and MAL9 heterologously expressed in Escherichia coli confirmed the correctness of the prediction—these proteins were verified promiscuous maltase-isomaltases. α-Glucosidases of earlier diverged yeasts L. starkeyi, B. adeninivorans and S. pombe showed sequence relatedness with α-glucosidases of filamentous fungi and bacilli.

Activity of the α-glucoside transporter Agt1 in Saccharomyces cerevisiae cells during dehydration-rehydration events

Microbial cells can enter a state of anhydrobiosis under desiccating conditions. One of the main determinants of viability during dehydration-rehydration cycles is structural integrity of the plasma membrane. Whereas much is known about phase transitions of the lipid bilayer, there is a paucity of information on changes in activity of plasma membrane proteins during dehydration-rehydration events. We selected the α-glucoside transporter Agt1 to gain insights into stress mechanisms/responses and ecophysiology during anhydrobiosis. As intracellular water content of S. cerevisiae strain 14 (a strain with moderate tolerance to dehydration-rehydration) was reduced to 1.5 g water/g dry weight, the activity of the Agt1 transporter decreased by 10–15 %. This indicates that functionality of this trans-membrane and relatively hydrophobic protein depends on water. Notably, however, levels of cell viability were retained. Prior incubation in the stress protectant xylitol increased stability of the plasma membrane but not Agt1. Studies were carried out using a comparator yeast which was highly resistant to dehydration-rehydration (S. cerevisiae strain 77). By contrast to S. cerevisiae strain 14, there was no significant reduction of Agt1 activity in S. cerevisiae strain 77 cells. These findings have implications for the ecophysiology of S. cerevisiae strains in natural and industrial systems.

Inhibition of Saccharomyces cerevisiae growth by simultaneous uptake of glucose and maltose

Saccharomyces cerevisiae expresses α-glucoside transporters, such as MalX1p (X=1(Agt1p), 2, 3, 4, and 6), which are proton symporters. These transporters are regulated at transcriptional and posttranslational levels in the presence of glucose. Malt wort contains glucose, maltose, and maltotriose, and the assimilation of maltose is delayed as a function of glucose concentration. With the objective of increasing beer fermentation rates, we characterized α-glucoside transporters and bred laboratory yeasts that expressed various α-glucoside transporters for the simultaneous uptake of different sugars. Mal21p was found to be the most resistant transporter to glucose-induced degradation, and strain (HD17) expressing MAL21 grew on a medium containing glucose or maltose, but not on a medium containing both sugars (YPDM). This unexpected growth defect was observed on a medium containing glucose and >0.1% maltose but was not exhibited by a strain that constitutively expressed maltase. The defect depended on intracellular maltose concentration. Although maltose accumulation caused a surge in turgor pressure, addition of sorbitol to YPDM did not increase growth. When strain HD17 was cultivated in a medium containing only maltose, protein synthesis was inhibited at early times but subsequently resumed with reduction in accumulated maltose, but not if the medium was exchanged for YPDM. We conclude that protein synthesis was terminated under the accumulation of maltose, regardless of extracellular osmolarity, and HD17 could not resume growth, because the intracellular concentration of maltose did not decrease due to insufficient synthesis of maltase. Yeast should incorporate maltose after expressing adequate maltase in beer brewing.

Specific Glucoside Transporters Influence Septal Structure and Function in the Filamentous, Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120.

When deprived of combined nitrogen, some filamentous cyanobacteria contain two cell types: vegetative cells that fix CO2 through oxygenic photosynthesis and heterocysts that are specialized in N2 fixation. In the diazotrophic filament, the vegetative cells provide the heterocysts with reduced carbon (mainly in the form of sucrose) and heterocysts provide the vegetative cells with combined nitrogen. Septal junctions traverse peptidoglycan through structures known as nanopores and appear to mediate intercellular molecular transfer that can be traced with fluorescent markers, including the sucrose analog esculin (a coumarin glucoside) that is incorporated into the cells. Uptake of esculin by the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 was inhibited by the α-glucosides sucrose and maltose. Analysis of Anabaena mutants identified components of three glucoside transporters that move esculin into the cells: GlsC (Alr4781) and GlsP (All0261) are an ATP-binding subunit and a permease subunit of two different ABC transporters, respectively, and HepP (All1711) is a major facilitator superfamily (MFS) protein that was shown previously to be involved in formation of the heterocyst envelope. Transfer of fluorescent markers (especially calcein) between vegetative cells of Anabaena was impaired by mutation of glucoside transporter genes. GlsP and HepP interact in bacterial two-hybrid assays with the septal junction-related protein SepJ, and GlsC was found to be necessary for the formation of a normal number of septal peptidoglycan nanopores and for normal subcellular localization of SepJ. Therefore, beyond their possible role in nutrient uptake in Anabaena, glucoside transporters influence the structure and function of septal junctions.IMPORTANCE Heterocyst-forming cyanobacteria have the ability to perform oxygenic photosynthesis and to assimilate atmospheric CO2 and N2 These organisms grow as filaments that fix these gases specifically in vegetative cells and heterocysts, respectively. For the filaments to grow, these types of cells exchange nutrients, including sucrose, which serves as a source of reducing power and of carbon skeletons for the heterocysts. Movement of sucrose between cells in the filament takes place through septal junctions and has been traced with a fluorescent sucrose analog, esculin, that can be taken up by the cells. Here, we identified α-glucoside transporters of Anabaena that mediate uptake of esculin and, notably, influence septal structure and the function of septal junctions.

Expression of Saccharomyces cerevisiae α-glucoside transporters under different growth conditions

Important biotechnological processes depend on the efficient fermentation by Saccharomyces cerevisiae yeasts of starch hydrolysates rich in maltose and maltotriose. The rate-limiting step for fermentation of these α-glucosides is the transport across the plasma membrane of the cells. In order to contribute to a better understanding of maltose and maltotriose metabolism by S. cerevisiae, we analyzed the expression of the main α glucoside transporter genes in two different yeast strains grown on media with glucose, maltose or maltotriose as carbon source. Although both yeast strains have higher αglucoside transport activity during growth on maltotriose, our results show similar expression levels of the analyzed genes on either maltose or maltrotriose media. Thus, our results indicate that, although the transport capacity of maltotriose grown cells is higher than that of maltose grown cells, maltotriose cannot be considered a better inducer of αglucoside transporter genes.

A putative α-glucoside transporter gene BbAGT1 contributes to carbohydrate utilization, growth, conidiation and virulence of filamentous entomopathogenic fungus Beauveria bassiana

Carbohydrate transporters are critical players mediating nutrient uptake during saprophytic and pathogenic growth for most filamentous fungi. For entomopathogenic fungi, such as Beauveria bassiana, assimilation of α-glucosides, in particular, trehalose, the major carbohydrate constituent of the insect haemolymph, has been hypothesized to represent an important ability for infectious growth within the insect hemocoel. In this study, a B. bassiana α-glucoside transporter homolog was identified and genetically characterized via generation of a targeted gene disruption mutant. Trehalose utilization was compromised in the mutant strain. In addition, inactivation of the α-glucoside transporter resulted in decreased conidial germination, growth, and yield on various carbohydrates (α-glucosides, monosaccharides and polyols) as compared to the wild-type strain. Insect bioassays revealed decreased mean lethal mortality time using both topical and intrahemocoel injection assays, although final mortality levels were comparable in both the mutant and wild type. Gene expression profiles showed altered expression of other putative transporters in the knockout mutant as compared to the wild type. These results highlighted complex sugar utilization and responsiveness in B. bassiana and the potential role for trehalose assimilation during fungal pathogenesis of insects.

Identification of Amino Acids Important for Substrate Specificity in Sucrose Transporters Using Gene Shuffling

Plant sucrose transporters (SUTs) are H(+)-coupled uptake transporters. Type I and II (SUTs) are phylogenetically related but have different substrate specificities. Type I SUTs transport sucrose, maltose, and a wide range of natural and synthetic α- and β-glucosides. Type II SUTs are more selective for sucrose and maltose. Here, we investigated the structural basis for this difference in substrate specificity. We used a novel gene shuffling method called synthetic template shuffling to introduce 62 differentially conserved amino acid residues from type I SUTs into OsSUT1, a type II SUT from rice. The OsSUT1 variants were tested for their ability to transport the fluorescent coumarin β-glucoside esculin when expressed in yeast. Fluorescent yeast cells were selected using fluorescence-activated cell sorting (FACS). Substitution of five amino acids present in type I SUTs in OsSUT1 was found to be sufficient to confer esculin uptake activity. The changes clustered in two areas of the OsSUT1 protein: in the first loop and the top of TMS2 (T80L and A86K) and in TMS5 (S220A, S221A, and T224Y). The substrate specificity of this OsSUT1 variant was almost identical to that of type I SUTs. Corresponding changes in the sugarcane type II transporter ShSUT1 also changed substrate specificity, indicating that these residues contribute to substrate specificity in type II SUTs in general.

Characterization of the Saccharomyces bayanus-type AGT1 transporter of lager yeast

Transport of maltose and maltotriose into the yeast cell is thought to be rate-limiting in the utilization of these sugars. The maltose and maltotriose transporters Malx1, Agt1, Mtt1 and Mphx are present in different combinations in brewer's yeast strains, conferring different maltose and maltotriose transport characteristics on the strains. A new putative maltose/maltotriose transporter ORF was identified during whole genome sequencing of the lager strain WS34/70 (Y. Nakao et al., DNA Res., 2009, 16, 115–129). Sequence comparisons suggested that this putative α-glucoside transporter might be a Saccharomyces bayanus counterpart of the Agt1 (Saccharomyces cerevisiae type) transporter. In the present work, the transporter coded by a SbAGT1 gene from a lager strain, A15 (and with the same sequence as the corresponding gene in WS34/70) was characterized. It is shown that this SbAGT1 encodes a functional α-glucoside transporter with a wide-substrate range, including maltose and maltotriose. Trehalose, α-methylglucoside and sucrose were inhibitors, suggesting they are also substrates. The SbAgt1 transporter had similar affinities for maltose and maltotriose (17 ± 7 and 22 ± 2 m m, respectively) and a higher Vmax for maltose than maltotriose (21 ± 7 and 12 ± 2 µmol min−1 g dry yeast−1, respectively). Copyright © 2012 The Institute of Brewing & Distilling

Identification of regulatory elements in the AGT1 promoter of ale and lager strains of brewer's yeast

Agt1 is an interesting α-glucoside transporter for the brewing industry, as it efficiently transports maltotriose, a sugar often remaining partly unused during beer fermentation. It has been shown that on maltose the expression level of AGT1 is much higher in ale strains than in lager strains, and that glucose represses the expression, particularly in the ale strains. In the present study the regulatory elements of the AGT1 promoter of one ale and two lager strains were identified by computational methods. Promoter regions up to 1.9 kbp upstream of the AGT1 gene were sequenced from the three brewer's yeast strains and the laboratory yeast strain CEN.PK-1D. The promoter sequence of the laboratory strain was identical to the AGT1 promoter of strain S288c of the Saccharomyces Genome Database, whereas the promoter sequences of the industrial strains diverged markedly from the S288c strain. The AGT1 promoter regions of the ale and lager strains were for the most part identical to each other, except for one 22 bp deletion and two 94 and 95 bp insertions in the ale strain. Computational analyses of promoter elements revealed that the promoter sequences contained several Mig1- and MAL-activator binding sites, as was expected. However, some of the Mig1 and MAL-activator binding sites were located on the two insertions of the ale strain, and thus offered a plausible explanation for the different expression pattern of the AGT1 gene in the ale strains. Accordingly, functional analysis of A60 ale and A15 lager strain AGT1 promoters fused to GFP (encoding the green fluorescent protein) showed a significant difference in the ability of these two promoters to drive GFP expression. Under the control of the AGT1 promoter of the ale strain the emergence of GFP was strongly induced by maltose, whereas only a low level of GFP was detected with the construct carrying the AGT1 promoter of the lager strain. Thus, the extra MAL-activator binding element, present in the AGT1 promoter of the ale strain, appears to be necessary to reach a high level of induction by maltose. Both AGT1 promoters were repressed by glucose but their derepression was different, possibly due to a distinct distribution of Mig1 elements in these two promoters.

Characterization of a New Multigene Family Encoding Isomaltases in the Yeast Saccharomyces cerevisiae, the IMA Family

It has been known for a long time that the yeast Saccharomyces cerevisiae can assimilate alpha-methylglucopyranoside and isomaltose. We here report the identification of 5 genes (YGR287c, YIL172c, YJL216c, YJL221c and YOL157c), which, similar to the SUCx, MALx, or HXTx multigene families, are located in the subtelomeric regions of different chromosomes. They share high nucleotide sequence identities between themselves (66-100%) and with the MALx2 genes (63-74%). Comparison of their amino acid sequences underlined a substitution of threonine by valine in region II, one of the four highly conserved regions of the alpha-glucosidase family. This change was previously shown to be sufficient to discriminate alpha-1,4- to alpha-1,6-glucosidase activity in YGR287c (Yamamoto, K., Nakayama, A., Yamamoto, Y., and Tabata, S. (2004) Eur. J. Biochem. 271, 3414-3420). We showed that each of these five genes encodes a protein with alpha-glucosidase activity on isomaltose, and we therefore renamed these genes IMA1 to IMA5 for IsoMAltase. Our results also illustrated that sequence polymorphisms among this family led to interesting variability of gene expression patterns and of catalytic efficiencies on different substrates, which altogether should account for the absence of functional redundancy for growth on isomaltose. Indeed, deletion studies revealed that IMA1/YGR287c encodes the major isomaltase and that growth on isomaltose required the presence of AGT1, which encodes an alpha-glucoside transporter. Expressions of IMA1 and IMA5/YJL216c were strongly induced by maltose, isomaltose, and alpha-methylglucopyranoside, in accordance with their regulation by the Malx3p-transcription system. The physiological relevance of this IMAx multigene family in S. cerevisiae is discussed.

Improved Fermentation Performance of a Lager Yeast after Repair of Its AGT1 Maltose and Maltotriose Transporter Genes

The use of more concentrated, so-called high-gravity and very-high-gravity (VHG) brewer's worts for the manufacture of beer has economic and environmental advantages. However, many current strains of brewer's yeasts ferment VHG worts slowly and incompletely, leaving undesirably large amounts of maltose and especially maltotriose in the final beers. alpha-Glucosides are transported into Saccharomyces yeasts by several transporters, including Agt1, which is a good carrier of both maltose and maltotriose. The AGT1 genes of brewer's ale yeast strains encode functional transporters, but the AGT1 genes of the lager strains studied contain a premature stop codon and do not encode functional transporters. In the present work, one or more copies of the AGT1 gene of a lager strain were repaired with DNA sequence from an ale strain and put under the control of a constitutive promoter. Compared to the untransformed strain, the transformants with repaired AGT1 had higher maltose transport activity, especially after growth on glucose (which represses endogenous alpha-glucoside transporter genes) and higher ratios of maltotriose transport activity to maltose transport activity. They fermented VHG (24 degrees Plato) wort faster and more completely, producing beers containing more ethanol and less residual maltose and maltotriose. The growth and sedimentation behaviors of the transformants were similar to those of the untransformed strain, as were the profiles of yeast-derived volatile aroma compounds in the beers.

Molecular Analysis of Maltotriose Active Transport and Fermentation by Saccharomyces cerevisiae Reveals a Determinant Role for the AGT1 Permease

Incomplete and/or sluggish maltotriose fermentation causes both quality and economic problems in the ale-brewing industry. Although it has been proposed previously that the sugar uptake must be responsible for these undesirable phenotypes, there have been conflicting reports on whether all the known alpha-glucoside transporters in Saccharomyces cerevisiae (MALx1, AGT1, and MPH2 and MPH3 transporters) allow efficient maltotriose utilization by yeast cells. We characterized the kinetics of yeast cell growth, sugar consumption, and ethanol production during maltose or maltotriose utilization by several S. cerevisiae yeast strains (both MAL constitutive and MAL inducible) and by their isogenic counterparts with specific deletions of the AGT1 gene. Our results clearly showed that yeast strains carrying functional permeases encoded by the MAL21, MAL31, and/or MAL41 gene in their plasma membranes were unable to utilize maltotriose. While both high- and low-affinity transport activities were responsible for maltose uptake from the medium, in the case of maltotriose, the only low-affinity (K(m), 36 +/- 2 mM) transport activity was mediated by the AGT1 permease. In conclusion, the AGT1 transporter is required for efficient maltotriose fermentation by S. cerevisiae yeasts, highlighting the importance of this permease for breeding and/or selection programs aimed at improving sluggish maltotriose fermentations.

Maltose and Maltotriose Active Transport and Fermentation by Saccharomyces Cerevisiaes

In the current study we analyzed the kinetics of growth, sugar consumption, and ethanol production by a Saccharomyces cerevisiae strain with the AGT1 permease as its unique α-glucoside transporter on synthetic or rich medium containing maltose or maltotriose. Our results show that to efficiently consume and ferment these sugars from the medium requires constitutive expression of the transporter gene, which can be achieved by the presence of a constitutive MAL (e.g., MAL63c) regulator. In addition to constitutive expression of the permease, growth on rich medium also increased the fermentation performance of the cells. Although a good correlation between the activity of the AGT1 transporter and the fermentation performance of the cells was observed, the same was not true for the activity of the intracellular α-glucosidase. The AGT1-containing S. cerevisiae strain fermented maltotriose more efficiently than maltose due to higher expression of the AGT1 permease during growth on maltotriose, leading to higher uptake rates of this sugar into the cells. Our results also indicate that during maltotriose fermentation by yeasts with constitutive expression of the AGT1 permease transport across the plasma membrane may no longer limit sugar utilization by S. cerevisiae cells.

Differences amongAGT1-encoded α-glucoside transporters and their ability to transport maltotriose inSaccharomyces yeasts

Improved fermentation of starch and its dextrin products would benefit the brewing and whiskey industries. Most strains ofSaccharomyces ferment glucose and maltose and partially ferment maltotriose, but are unable to utilise the larger dextrin products of starch. This utilisation pattern is partly attributed to the ability of yeast cells to transport the aforementioned mono-, di- and trisaccharides into the cytosol. The maltotriose transporting efficiency varies between differentSaccharomyces strains. In this study, severalSaccharomyces strains, including whiskey strains, were screened for growth on maltotriose. TheAGT1 genes, which encode a maltose transporter that show affinity for maltotriose uptake, were isolated from the strains that grew strongest in media with maltotriose as sole carbon source. The isolatedAGT1 alleles were sequenced and their chromosomal locations determined in the strains from which they were cloned. Nucleotide and deduced amino acid sequences of the isolated genes shared 95% and 98% identity, respectively. The efficiency of maltotriose transport was determined by expressing theAGT1 variants in an identical genetic background. TheK m values obtained for all the permeases were very similar (≈3), but the permease with improved performance for maltotriose transport showed an approximately 30% higherV max value than for the others. The data obtained suggest that the genetic variation among theAGT1-encoded transporters is reason for the variation in maltotriose transport efficiency among differentSaccharomyces strains. This study offers prospects for the development of yeast strains with improved maltose and maltotriose uptake capabilities that, in turn, could increase the overall fermentation efficiencies in the beer and whiskey industries.

Maltotriose Utilization by Industrial Saccharomyces Strains: Characterization of a New Member of the α-Glucoside Transporter Family

Maltotriose utilization by Saccharomyces cerevisiae and closely related yeasts is important to industrial processes based on starch hydrolysates, where the trisaccharide is present in significant concentrations and often is not completely consumed. We undertook an integrated study to better understand maltotriose metabolism in a mixture with glucose and maltose. Physiological data obtained for a particularly fast-growing distiller's strain (PYCC 5297) showed that, in contrast to what has been previously reported for other strains, maltotriose is essentially fermented. The respiratory quotient was, however, considerably higher for maltotriose (0.36) than for maltose (0.16) or glucose (0.11). To assess the role of transport in the sequential utilization of maltose and maltotriose, we investigated the presence of genes involved in maltotriose uptake in the type strain of Saccharomyces carlsbergensis (PYCC 4457). To this end, a previously constructed genomic library was used to identify maltotriose transporter genes by functional complementation of a strain devoid of known maltose transporters. One gene, clearly belonging to the MAL transporter family, was repeatedly isolated from the library. Sequence comparison showed that the novel gene (designated MTY1) shares 90% and 54% identity with MAL31 and AGT1, respectively. However, expression of Mty1p restores growth of the S. cerevisiae receptor strain on both maltose and maltotriose, whereas the closely related Mal31p supports growth on maltose only and Agt1p supports growth on a wider range of substrates, including maltose and maltotriose. Interestingly, Mty1p displays higher affinity for maltotriose than for maltose, a new feature among all the alpha-glucoside transporters described so far.

Trehalose reserve in Saccharomyces cerevisiae: phenomenon of transport, accumulation and role in cell viability

Strains of Saccharomyces cerevisiae deleted for TPS1 encoding trehalose-6-phosphate synthase still accumulate trehalose when harbouring a functional MAL locus. We demonstrate that this accumulation results from an active uptake of trehalose present in the ‘yeast extract’ used to make the enriched culture media and that no accumulation is observed in mineral media. The uptake of trehalose was shown to be mediated by the α-glucoside transporter encoded by AGT1, the expression of which is linked to the presence of a functional MAL locus. Deletion of this gene in a MAL + tps1 mutant abolished trehalose accumulation on a maltose or galactose mineral medium. However, small amounts of disaccharide were still detected in a agt1 tps1 double mutant when the medium was supplemented with 10 g trehalose l −1, indicating the existence of a non-concentrative low-affinity sugar transporter. The presence of the high-affinity trehalose permease allowed us to investigate the effect of increasing exogenous trehalose from 0 to 10 g l −1 on intracellular accumulation. A maximum of ca. 10% (wt/wt dry cells) trehalose was attained in the presence of only 1 g l −1 of disaccharide in the medium. The capability to monitor the intracellular content of trehalose by varying its extracellular concentration, independent of genetic alterations of the trehalose metabolic machinery, allowed the remarkable contribution of this molecule in stress tolerance to be demonstrated, as the higher the trehalose content, the longer the cell survival to a severe heat shock and to glucose starvation.

Kinetics of active sucrose transport in Saccharomyces cerevisiae

The kinetic analysis of active sucrose-H + uptake by Saccharomyces cerevisiae revealed the presence of two transport systems with high and low affinity for sucrose. The MAL2T permease has a low affinity ( K m = 120 ±20 mM) for sucrose, while the α-glucoside transporter encoded by the AGT1 gene is a high affinity sucrose-H + symporter ( K m = 7.9±0.8 mM) that increases the specific growth rate of cells growing on sucrose.