Microarray karyotyping of maltose‐fermenting Saccharomyces yeasts with differing maltotriose utilization profiles reveals copy number variation in genes involved in maltose and maltotriose utilization

Aims: We performed an analysis of maltotriose utilization by 52 Saccharomyces yeast strains able to ferment maltose efficiently and correlated the observed phenotypes with differences in the copy number of genes possibly involved in maltotriose utilization by yeast cells. Methods and Results: The analysis of maltose and maltotriose utilization by laboratory and industrial strains of the species Saccharomyces cerevisiae and Saccharomyces pastorianus (a natural S. cerevisiae/Saccharomyces bayanus hybrid) was carried out using microscale liquid cultivation, as well as in aerobic batch cultures. All strains utilize maltose efficiently as a carbon source, but three different phenotypes were observed for maltotriose utilization: efficient growth, slow/delayed growth and no growth. Through microarray karyotyping and pulsed‐field gel electrophoresis blots, we analysed the copy number and localization of several maltose‐related genes in selected S. cerevisiae strains. While most strains lacked the MPH2 and MPH3 transporter genes, almost all strains analysed had the AGT1 gene and increased copy number of MALx1 permeases. Conclusions: Our results showed that S. pastorianus yeast strains utilized maltotriose more efficiently than S. cerevisiae strains and highlighted the importance of the AGT1 gene for efficient maltotriose utilization by S. cerevisiae yeasts. Significance and Impact of the Study: Our results revealed new maltotriose utilization phenotypes, contributing to a better understanding of the metabolism of this carbon source for improved fermentation by Saccharomyces yeasts.     

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 α-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.     

A Large Set of Newly Created Interspecific Saccharomyces Hybrids Increases Aromatic Diversity in Lager Beers

Lager beer is the most consumed alcoholic beverage in the world. Its production process is marked by a fermentation conducted at low (8 to 15°C) temperatures and by the use of Saccharomyces pastorianus, an interspecific hybrid between Saccharomyces cerevisiae and the cold-tolerant Saccharomyces eubayanus. Recent whole-genome-sequencing efforts revealed that the currently available lager yeasts belong to one of only two archetypes, “Saaz” and “Frohberg.” This limited genetic variation likely reflects that all lager yeasts descend from only two separate interspecific hybridization events, which may also explain the relatively limited aromatic diversity between the available lager beer yeasts compared to, for example, wine and ale beer yeasts. In this study, 31 novel interspecific yeast hybrids were developed, resulting from large-scale robot-assisted selection and breeding between carefully selected strains of S. cerevisiae (six strains) and S. eubayanus (two strains). Interestingly, many of the resulting hybrids showed a broader temperature tolerance than their parental strains and reference S. pastorianus yeasts. Moreover, they combined a high fermentation capacity with a desirable aroma profile in laboratory-scale lager beer fermentations, thereby successfully enriching the currently available lager yeast biodiversity. Pilot-scale trials further confirmed the industrial potential of these hybrids and identified one strain, hybrid H29, which combines a fast fermentation, high attenuation, and the production of a complex, desirable fruity aroma.     

Brewing up a storm: The genomes of lager yeasts and how they evolved

Yeasts used in the production of lager beers belong to the species Saccharomyces pastorianus, an interspecies hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus. The hybridisation event happened approximately 500–600 years ago and therefore S. pastorianus may be considered as a newly evolving species. The happenstance of the hybridisation event created a novel species, with unique genetic characteristics, ideal for the fermentation of sugars to produce flavoursome beer. Lager yeast strains retain the chromosomes of both parental species and also have sets of novel hybrid chromosomes that arose by recombination between the homeologous parental chromosomes. The lager yeasts are subdivided into two groups (I and II) based on the S. cerevisiae: S. eubayanus gene content and the types and numbers of hybrid chromosomes. Recently, whole genome sequences for several Group I and II lager yeasts and for many S. cerevisiae and S. eubayanus isolates have become available. Here we review the available genome data and discuss the likely origins of the parental species that gave rise to S. pastorianus. We review the compiled data on the composition of the lager yeast genomes and consider several evolutionary models to account for the emergence of the two distinct types of lager yeasts.     

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 α-glucoside transporters described so far.     

Improvement of maltotriose fermentation by Saccharomyces cerevisiae

AIMS: To enhance the fermentation of maltotriose by industrial Saccharomyces cerevisiae strains. METHODS AND RESULTS: The capability to ferment maltotriose by an industrial yeast strain that uses this sugar aerobically was tested in shake flasks containing rich medium. While the presence of maltose in the medium did not improve maltotriose fermentation, enhanced and constitutive expression of the AGT1 permease not only increased the uptake of maltotriose, but allowed efficient maltotriose fermentation by this strain. Supplementation of the growth medium with 20 mmol magnesium l(-1) also increased maltotriose fermentation. CONCLUSIONS: Over expression of the AGT1 permease and magnesium supplementation improved maltotriose fermentation by an industrial yeast strain that respired but did not ferment this sugar. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes to the elucidation of the roles of the AGT1 permease and nutrients in the fermentation of all sugars present in starch hydrolysates, a highly desirable trait for several industrial yeasts.     

The Thr505 and Ser557 residues of the AGT1-encoded alpha-glucoside transporter are critical for maltotriose transport in Saccharomyces cerevisiae

Aims:  The main objective of this study was to identify amino acid residues in the AGT1‐encoded α‐glucoside transporter (Agt1p) that are critical for efficient transport of maltotriose in the yeast Saccharomyces cerevisiae. Methods and Results:  The sequences of two AGT1‐encoded α‐glucoside transporters with different efficiencies of maltotriose transport in two Saccharomyces strains (WH310 and WH314) were compared. The sequence variations and discrepancies between these two proteins (Agt1p_WH310 and Agt1p_WH314) were investigated for potential effects on the functionality and maltotriose transport efficiency of these two AGT1‐encoded α‐glucoside transporters. A 23‐amino‐acid C‐terminal truncation proved not to be critical for maltotriose affinity. The identification of three amino acid differences, which potentially could have been instrumental in the transportation of maltotriose, were further investigated. Single mutations were created to restore the point mutations I505T, V549A and T557S one by one. The single site mutant V549A showed a decrease in maltotriose transport ability, and the I505T and T557S mutants showed complete reduction in maltotriose transport. Conclusions:  The amino acids Thr⁵⁰⁵ and Ser⁵⁵⁷, which are respectively located in the transmembrane (TM) segment TM¹¹ and on the intracellular segment after TM¹² of the AGT1‐encoded α‐glucoside transporters, are critical for efficient transport of maltotriose in S. cerevisiae. Significance and Impact of the Study:  Improved fermentation of starch and its dextrin products, such as maltotriose and maltose, would benefit the brewing and whisky industries. This study could facilitate the development of engineered maltotriose transporters adapted to starch‐efficient fermentation systems, and 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 whisky industries.     

Maltotriose utilization by industrial Saccharomyces strains: characterization of a new member of the alpha-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.     

Population structure and reticulate evolution of Saccharomyces eubayanus and its lager‐brewing hybrids

Reticulate evolution can be a major driver of diversification into new niches, especially in disturbed habitats and at the edges of ranges. Industrial fermentation strains of yeast provide a window into these processes, but progress has been hampered by a limited understanding of the natural diversity and distribution of Saccharomyces species and populations. For example, lager beer is brewed with Saccharomyces pastorianus, an alloploid hybrid of S. cerevisiae and S. eubayanus, a species only recently discovered in Patagonia, Argentina. Here, we report that genetically diverse strains of S. eubayanus are readily isolated from Patagonia, demonstrating that the species is well established there. Analyses of multilocus sequence data strongly suggest that there are two diverse and highly differentiated Patagonian populations. The low nucleotide diversity found in the S. eubayanus moiety of hybrid European brewing strains suggests that their alleles were drawn from a small subpopulation that is closely related to one of the Patagonian populations. For the first time, we also report the rare isolation of S. eubayanus outside Patagonia, in Wisconsin, USA. In contrast to the clear population differentiation in Patagonia, the North American strains represent a recent and possibly transient admixture of the two Patagonian populations. These complex and varied reticulation events are not adequately captured by conventional phylogenetic methods and required analyses of Bayesian concordance factors and phylogenetic networks to accurately summarize and interpret. These findings show how genetically diverse eukaryotic microbes can produce rare but economically important hybrids with low genetic diversity when they migrate from their natural ecological context.     

Chromosomal Copy Number Variation in Saccharomyces pastorianus Is Evidence for Extensive Genome Dynamics in Industrial Lager Brewing Strains

Lager brewing strains of Saccharomyces pastorianus are natural interspecific hybrids originating from the spontaneous hybridization of Saccharomyces cerevisiae and Saccharomyces eubayanus. Over the past 500 years, S. pastorianus has been domesticated to become one of the most important industrial microorganisms. Production of lager-type beers requires a set of essential phenotypes, including the ability to ferment maltose and maltotriose at low temperature, the production of flavors and aromas, and the ability to flocculate. Understanding of the molecular basis of complex brewing-related phenotypic traits is a prerequisite for rational strain improvement. While genome sequences have been reported, the variability and dynamics of S. pastorianus genomes have not been investigated in detail. Here, using deep sequencing and chromosome copy number analysis, we showed that S. pastorianus strain CBS1483 exhibited extensive aneuploidy. This was confirmed by quantitative PCR and by flow cytometry. As a direct consequence of this aneuploidy, a massive number of sequence variants was identified, leading to at least 1,800 additional protein variants in S. pastorianus CBS1483. Analysis of eight additional S. pastorianus strains revealed that the previously defined group I strains showed comparable karyotypes, while group II strains showed large interstrain karyotypic variability. Comparison of three strains with nearly identical genome sequences revealed substantial chromosome copy number variation, which may contribute to strain-specific phenotypic traits. The observed variability of lager yeast genomes demonstrates that systematic linking of genotype to phenotype requires a three-dimensional genome analysis encompassing physical chromosomal structures, the copy number of individual chromosomes or chromosomal regions, and the allelic variation of copies of individual genes.     

Maltotriose metabolism by Saccharomyces cerevisiae

Saccharomyces cerevisiae grew slower but reached higher cellular densities when grown on 20 g maltotriose l^–1 than on the same concentration of glucose or maltose. Antimycin A (3 mg l^–1) prevented growth on maltotriose, but not on glucose or maltose, indicating that it is not fermented but is degraded aerobically. This was confirmed by the absence of ethanol and glycerol production. Active uptake of maltotriose across the plasma membrane is the limiting step for metabolism, and the low rate of maltotriose transport observed in maltotriose-grown cells is probably one of the main reasons for the absence of maltotriose fermentation by S. cerevisiae cells.     

Characterization of AGT1 encoding a general α-glucoside transporter from Saccharomyces

Molecular genetic analysis is used to characterize the AGT1 gene encoding an α-glucoside transporter. AGT1 is found in many Saccharomyces cerevisiae laboratory strains and maps to a naturally occurring, partially functional allele of the MAL1 locus. Agt1p is a highly hydrophobic, postulated integral membrane protein. It is 57% identical to Mal61p, the maltose permease encoded at MAL6 , and is also a member of the 12 transmembrane domain superfamily of sugar transporters. Like Mal61p, Agt1p is a high-affinity, maltose/proton symporter, but Mal61p is capable of transporting only maltose and turanose, while Agt1p transports these two α-glucosides as well as several others including isomaltose, α-methylglucoside, maltotriose, palatinose, trehalose and melezitose. AGT1 expression is maltose inducible and induction is mediated by the Mal-activator. The sequence of the upstream region of AGT1 is identical to that of the maltose-inducible MAL61 gene over a 469 bp region containing the UAS_MAL but the 315 bp sequence immediately upstream of AGT1 shows no significant homology to the sequence immediately upstream of MAL61 . The evolutionary origin of the MAL1 allele to which AGT1 maps and the relationship of AGT1 to other α-glucoside fermentation genes 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. α-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 α-glucoside transporter genes) and higher ratios of maltotriose transport activity to maltose transport activity. They fermented VHG (24° 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.The main fermentable sugars in brewer''s wort are maltose (ca. 60% of the total), maltotriose (ca. 25%), and glucose (ca. 15%). In traditional brewery fermentations, worts of about 11° Plato (°P) are used, corresponding to a total fermentable sugar concentration of about 80 g · liter⁻¹. Many modern breweries ferment high-gravity worts (15 to 17°P), and there are efforts to raise the concentration to 25°P, corresponding to a total sugar concentration of about 200 g · liter⁻¹. Industrial use of such very-high-gravity (VHG) worts is attractive because it offers increased production capacity from the same-size brew house and fermentation facilities, decreased energy consumption, and decreased labor, cleaning, and effluent costs (34, 35).Whereas glucose, which is used first, is transported into yeast cells by facilitated diffusion, the α-glucosides maltose and maltotriose are carried by proton symporters (2, 26, 39). Maltose transport seems to have a high level of control over the fermentation rate. Thus, during the early and middle stages of fermentation of brewer''s wort by a lager yeast, the specific rate of maltose consumption was the same as the specific zero-trans maltose uptake rate measured off line with each day''s yeast in each day''s wort spiked with [¹⁴C]maltose (27). Furthermore, introducing a constitutive MAL61 (maltose transporter) gene into a brewer''s yeast on a multicopy plasmid accelerated the fermentation of high-gravity worts (17). Maltotriose is the last sugar to be used in brewing fermentations, and significant amounts of residual maltotriose sometimes remain in beer, causing economic losses (lower yield of ethanol on wort carbohydrate) and possibly undesirable organoleptic effects. The problem of residual sugars in beer is more serious when high-gravity and VHG worts are used. Some, but not all, maltose transporters can also carry maltotriose. The MALx1 genes (x = 1 to 4 and 6) encode transporters that carry maltose efficiently but are generally believed to have little or no activity toward maltotriose (1, 3, 13, 30), although substantial activity toward maltotriose was reported by Day et al. (4). Some yeast strains contain a gene 57% identical to MAL11 that is usually known as AGT1 but is recorded in the Saccharomyces Genome Database (SGDB) as MAL11. The Agt1 transporter has relatively high activity toward maltotriose, as well as maltose (13), and similar K_m values (4 to 5 mM) for these two substrates (4). Alves et al. (1) found that the specific deletion of AGT1 from several Saccharomyces cerevisiae strains also containing at least one MALx1 gene (MAL21, MAL31, and/or MAL41) abolished their ability to transport maltotriose but did not decrease their maltose transport activity. These results supported the belief that the Mal21, Mal31, and Mal41 transporters cannot carry maltotriose, though it remains possible that there are differences between Malx1 transporters from different strains. The same group has also shown (33) that overexpression of AGT1 on a multicopy plasmid in an industrial yeast strain with a very limited ability to ferment maltotriose provided the strain with increased maltotriose uptake activity and the ability to ferment maltotriose efficiently. In 2005, a novel kind of α-glucoside transporter was independently found by two groups (6, 30) in some industrial strains of brewer''s, baker''s, and distiller''s yeasts. These transporters are coded by MTT1 (also called MTY1) genes, which are 90 and 54% identical to the MAL31 and AGT1 genes, respectively. The Mtt1 transporters have high activity toward maltotriose and are the only known α-glucoside transporters with lower K_m values for maltotriose than for maltose (30).Before the discovery of the MTT1 genes, Vidgren et al. (36) sequenced AGT1 genes from two apparently unrelated lager strains and two apparently unrelated ale strains of brewer''s yeast. Surprisingly, at that time (because other maltotriose transporters were not known), the AGT1 genes from the lager strains contained an insertion of one nucleotide, resulting in a premature stop codon, and encoded a truncated, nonfunctional 394-amino-acid polypeptide, whereas those from the ale strains encoded full-length 616-amino-acid transporters. This premature stop codon was later shown (37) to be present in AGT1 genes from all eight of the lager strains tested but was not in any of the four ale strains tested, whereas MTT1 genes were present in all of the lager strains tested but in none of the ale strains tested.In the present work, we have tested whether lager fermentations can be accelerated and residual maltotriose levels decreased by repairing the defective AGT1 genes of lager strains with appropriate DNA sequences from ale strains. Furthermore, the MALx1 and AGT1 genes are repressed by glucose and induced by α-glucosides (9, 16, 19, 25), so that replacing the native AGT1 promoter with a constitutive S. cerevisiae promoter might also increase α-glucoside transport activity and accelerate wort fermentations. The objectives of the present work were to confirm that α-glucoside transport has a high level of control over the rate and extent of wort fermentation and to create a genetically modified lager yeast strain that has improved fermentation performance but contains only Saccharomyces DNA.     

Deciphering the Hybridisation History Leading to the Lager Lineage Based on the Mosaic Genomes of Saccharomyces bayanus Strains NBRC1948 and CBS380T

Saccharomyces bayanus is a yeast species described as one of the two parents of the hybrid brewing yeast S. pastorianus. Strains CBS380^T and NBRC1948 have been retained successively as pure-line representatives of S. bayanus. In the present study, sequence analyses confirmed and upgraded our previous finding: S. bayanus type strain CBS380^T harbours a mosaic genome. The genome of strain NBRC1948 was also revealed to be mosaic. Both genomes were characterized by amplification and sequencing of different markers, including genes involved in maltotriose utilization or genes detected by array-CGH mapping. Sequence comparisons with public Saccharomyces spp. nucleotide sequences revealed that the CBS380^T and NBRC1948 genomes are composed of: a predominant non-cerevisiae genetic background belonging to S. uvarum, a second unidentified species provisionally named S. lagerae, and several introgressed S. cerevisiae fragments. The largest cerevisiae-introgressed DNA common to both genomes totals 70kb in length and is distributed in three contigs, cA, cB and cC. These vary in terms of length and presence of MAL31 or MTY1 (maltotriose-transporter gene). In NBRC1948, two additional cerevisiae-contigs, cD and cE, totaling 12kb in length, as well as several smaller cerevisiae fragments were identified. All of these contigs were partially detected in the genomes of S. pastorianus lager strains CBS1503 (S. monacensis) and CBS1513 (S. carlsbergensis) explaining the noticeable common ability of S. bayanus and S. pastorianus to metabolize maltotriose. NBRC1948 was shown to be inter-fertile with S. uvarum CBS7001. The cross involving these two strains produced F1 segregants resembling the strains CBS380^T or NRRLY-1551. This demonstrates that these S. bayanus strains were the offspring of a cross between S. uvarum and a strain similar to NBRC1948. Phylogenies established with selected cerevisiae and non-cerevisiae genes allowed us to decipher the complex hybridisation events linking S. lagerae/S. uvarum/S. cerevisiae with their hybrid species, S. bayanus/pastorianus.     

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.     

Characterization and Functional Analysis of the MAL and MPH Loci for Maltose Utilization in Some Ale and Lager Yeast Strains

Maltose and maltotriose are the major sugars in brewer's wort. Brewer's yeasts contain multiple genes for maltose transporters. It is not known which of these express functional transporters. We correlated maltose transport kinetics with the genotypes of some ale and lager yeasts. Maltose transport by two ale strains was strongly inhibited by other α-glucosides, suggesting the use of broad substrate specificity transporters, such as Agt1p. Maltose transport by three lager strains was weakly inhibited by other α-glucosides, suggesting the use of narrow substrate specificity transporters. Hybridization studies showed that all five strains contained complete MAL1, MAL2, MAL3, and MAL4 loci, except for one ale strain, which lacked a MAL2 locus. All five strains also contained both AGT1 (coding a broad specificity α-glucoside transporter) and MAL11 alleles. MPH genes (maltose permease homologues) were present in the lager but not in the ale strains. During growth on maltose, the lager strains expressed AGT1 at low levels and MALx1 genes at high levels, whereas the ale strains expressed AGT1 at high levels and MALx1 genes at low levels. MPHx expression was negligible in all strains. The AGT1 sequences from the ale strains encoded full-length (616 amino acid) polypeptides, but those from both sequenced lager strains encoded truncated (394 amino acid) polypeptides that are unlikely to be functional transporters. Thus, despite the apparently similar genotypes of these ale and lager strains revealed by hybridization, maltose is predominantly carried by AGT1-encoded transporters in the ale strains and by MALx1-encoded transporters in the lager strains.     

Metabolic engineering for improved fermentation of pentoses by yeasts

The fermentation of xylose is essential for the bioconversion of lignocellulose to fuels and chemicals, but wild-type strains of Saccharomyces cerevisiae do not metabolize xylose, so researchers have engineered xylose metabolism in this yeast. Glucose transporters mediate xylose uptake, but no transporter specific for xylose has yet been identified. Over-expressing genes for aldose (xylose) reductase, xylitol dehydrogenase and moderate levels of xylulokinase enable xylose assimilation and fermentation, but a balanced supply of NAD(P) and NAD(P)H must be maintained to avoid xylitol production. Reducing production of NADPH by blocking the oxidative pentose phosphate cycle can reduce xylitol formation, but this occurs at the expense of xylose assimilation. Respiration is critical for growth on xylose by both native xylose-fermenting yeasts and recombinant S, cerevisiae. Anaerobic growth by recombinant mutants has been reported. Reducing the respiration capacity of xylose-metabolizing yeasts increases ethanol production. Recently, two routes for arabinose metabolism have been engineered in S. cerevisiae and adapted strains of Pichia stipitis have been shown to ferment hydrolysates with ethanol yields of 0.45 g g^–1 sugar consumed, so commercialization seems feasible for some applications.     

Characterization and functional analysis of the MAL and MPH Loci for maltose utilization in some ale and lager yeast strains

Maltose and maltotriose are the major sugars in brewer's wort. Brewer's yeasts contain multiple genes for maltose transporters. It is not known which of these express functional transporters. We correlated maltose transport kinetics with the genotypes of some ale and lager yeasts. Maltose transport by two ale strains was strongly inhibited by other alpha-glucosides, suggesting the use of broad substrate specificity transporters, such as Agt1p. Maltose transport by three lager strains was weakly inhibited by other alpha-glucosides, suggesting the use of narrow substrate specificity transporters. Hybridization studies showed that all five strains contained complete MAL1, MAL2, MAL3, and MAL4 loci, except for one ale strain, which lacked a MAL2 locus. All five strains also contained both AGT1 (coding a broad specificity alpha-glucoside transporter) and MAL11 alleles. MPH genes (maltose permease homologues) were present in the lager but not in the ale strains. During growth on maltose, the lager strains expressed AGT1 at low levels and MALx1 genes at high levels, whereas the ale strains expressed AGT1 at high levels and MALx1 genes at low levels. MPHx expression was negligible in all strains. The AGT1 sequences from the ale strains encoded full-length (616 amino acid) polypeptides, but those from both sequenced lager strains encoded truncated (394 amino acid) polypeptides that are unlikely to be functional transporters. Thus, despite the apparently similar genotypes of these ale and lager strains revealed by hybridization, maltose is predominantly carried by AGT1-encoded transporters in the ale strains and by MALx1-encoded transporters in the lager strains.