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Boris U. Stambuk - One of the best experts on this subject based on the ideXlab platform.
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key amino acid residues of the agt1 permease required for Maltotriose consumption and fermentation by saccharomyces cerevisiae
Journal of Applied Microbiology, 2019Co-Authors: P. S. De Araujo, Sérgio L. Alves, D. Trichez, Luiz Claudio Miletti, M M Knychala, Clara Figueiredo, M A A Da Silva, Boris U. StambukAbstract:Aims The AGT1 gene encodes for a general α-glucoside-H+ symporter required for efficient Maltotriose fermentation by Saccharomyces cerevisiae. In the present study, we analysed the involvement of four charged amino acid residues present in this transporter that are required for Maltotriose consumption and fermentation by yeast cells. Methods and results By using a knowledge-driven approach based on charge, conservation, location, three-dimensional (3D) structural modelling and molecular docking analysis, we identified four amino acid residues (Glu-120, Asp-123, Glu-167 and Arg-504) in the AGT1 permease that could mediate substrate binding and translocation. Mutant permeases were generated by site-directed mutagenesis of these charged residues, and expressed in a yeast strain lacking this permease (agt1∆). While mutating the Arg-504 or Glu-120 residues into alanine totally abolished (R504A mutant) or greatly reduced (E120A mutant) Maltotriose consumption by yeast cells, as well as impaired the active transport of several other α-glucosides, in the case of the Asp-123 and Glu-167 amino acids, it was necessary to mutate both residues (D123G/E167A mutant) in order to impair Maltotriose consumption and fermentation. Conclusions Based on the results obtained with mutant proteins, molecular docking and the localization of amino acid residues, we propose a transport mechanism for the AGT1 permease. Significance and impact of the study Our results present new insights into the structural basis for active α-glucoside-H+ symport activity by yeast transporters, providing the molecular bases for improving the catalytic properties of this type of sugar transporters.
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extracellular Maltotriose hydrolysis by saccharomyces cerevisiae cells lacking the agt1 permease
Letters in Applied Microbiology, 2018Co-Authors: Johan M Thevelein, Sérgio L. Alves, Boris U. StambukAbstract:In brewing, Maltotriose is the least preferred sugar for uptake by Saccharomyces cerevisiae cells. Although the AGT1 permease is required for efficient Maltotriose fermentation, we have described a new phenotype in some agt1Δ strains of which the cells do not grow on Maltotriose during the first 3-4 days of incubation, but after that, they start to grow on the sugar aerobically. Aiming to characterize this new phenotype, we performed microarray gene expression analysis which indicated upregulation of high-affinity glucose transporters (HXT4, HXT6 and HXT7) and α-glucosidases (MAL12 and IMA5) during this delayed cellular growth. Since these results suggested that this phenotype might be due to extracellular hydrolysis of Maltotriose, we attempted to detect glucose in the media during growth. When an hxt-null agt1Δ strain was grown on Maltotriose, it also showed the delayed growth on this carbon source, and glucose accumulated in the medium during Maltotriose consumption. Considering that the poorly characterized α-glucosidase encoded by IMA5 was among the overexpressed genes, we deleted this gene from an agt1Δ strain that showed delayed growth on Maltotriose. The ima5Δ agt1Δ strain showed no Maltotriose utilization even after 200 h of incubation, suggesting that IMA5 is likely responsible for the extracellular Maltotriose hydrolysis. SIGNIFICANCE AND IMPACT OF THE STUDY Maltotriose is the second most abundant sugar present in brewing. However, many yeast strains have difficulties to consume Maltotriose, mainly because of its low uptake rate by the yeast cells when compared to glucose and maltose uptake. The AGT1 permease is required for efficient Maltotriose fermentation, but some strains deleted in this gene are still able to grow on Maltotriose after an extensive lag phase. This manuscript shows that such delayed growth on Maltotriose is a consequence of extracellular hydrolysis of the sugar. Our results also indicate that the IMA5-encoded α-glucosidase is likely the enzyme responsible for this phenotype.
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efficient Maltotriose fermentation through hydrolysis mediated by the intracellular invertase of saccharomyces cerevisiae
BMC Proceedings, 2014Co-Authors: Victor Ribeiro De Godoy, Gabriela Muller, Boris U. StambukAbstract:Background It is well known that in the yeast S. cerevisiae the sugars sucrose and maltose/Maltotriose are metabolized by different pathways: sucrose is hydrolyzed by extracellular invertase (encoded by SUC genes), while maltose and Maltotriose are actively transported into the cell and hydrolyzed by intracellular a-glucosidases (both proteins encoded by the MAL genes). Nevertheless, several reports have shown that some SUC genes can be located proximal to MAL genes at the telomeres of different chromosomes. Furthermore, the SUC genes also allow the synthesis of an intracellular form of invertase, an enzyme with no obvious function in yeasts [1]. We have already shown that sucrose can be metabolized by yeast cells through MAL-encoded transporters and a-glucosidases. Methods, results and conclusions Now, our results will show that Maltotriose can be efficiently fermented by S. cerevisiae cells through its active transport mediated by the AGT1 permease, a MAL transporter required for Maltotriose utilization [2,3], and its intracellular hydrolysis mediated by the cytoplasmic invertase. The Brazilian industrial fuel-ethanol strain CAT-1 cannot ferment Maltotriose efficiently due to a defective promoter of the AGT1 gene [4]. To increase Maltotriose fermentation by this strain, we placed a strong promoter (PGPD )i n theAGT1 gene of strain CAT-1, generating strain GMY05. While the AGT1 gene was indeed overexpressed in this strain (measured by real-time PCR and a specific transport assay), Maltotriose was still not fermented efficiently. However, when we over-expressed the intracellular form of invertase, by replacing the signal sequence of the SUC2 gene with the strong PPGK promoter, the resulting iSUC2 strain GMY08 fermented Maltotriose efficiently. Using conditions were the MAL-encoded a-glucosidases would not be expressed, we could show that the intracellular form of invertase hydrolyzes Maltotriose efficiently (but not maltose or p-nitrophenyl-a-glucoside), specially at the cytoplasmic pH of 7.0. Under the same conditions we purified the intracellular invertase by ion-exchange chromatography, and the identity of the enzyme confirmed by mass spectrophotometry. With the purified enzyme we performed enzymatic tests that corroborated our previous analysis, showing that intracellular invertase hydrolyzes Maltotriose. Thus, our results indicate an unexpected overlap in sucrose-Maltotriose metabolism by yeast cells, showing that the intracellular invertase allows efficient Maltotriose hydrolysis, and offers new approaches that can be applied to optimize several industrial fermentation processes that use starch hydrolysates, including production of distilled beverages, brewing and backing.
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Characterization of Maltotriose transporters from the Saccharomyces eubayanus subgenome of the hybrid Saccharomyces pastorianus lager brewing yeast strain Weihenstephan 34/70
Letters in applied microbiology, 2012Co-Authors: F.e.m. Cousseau, Sérgio L. Alves, D. Trichez, Boris U. StambukAbstract:The genome from the Saccharomyces pastorianus industrial lager brewing strain Weihenstephan 34/70, a natural Saccharomyces cerevisiae/Saccharomyces eubayanus hybrid, indicated the presence of two different Maltotriose transporter genes: a new gene in the S. eubayanus subgenome with 81% of homology to the AGT1 permease from S. cerevisiae, and an amplification of the S. eubayanus MTY1 Maltotriose permease previously identified in S. pastorianus yeasts. To characterize these S. eubayanus transporter genes, we used a S. cerevisiae strain deleted in the AGT1 permease and introduced the desired permease gene(s) into this locus through homologous recombination. Our results indicate that both the MTY1 and AGT1 genes from the S. eubayanus subgenome encode functional Maltotriose transporters that allow fermentation of this sugar by yeast cells, despite their apparent differences in the kinetics of Maltotriose-H(+) symport activity. The presence of two Maltotriose transporters in the S. eubayanus subgenome not only highlights the importance of sugar transport for efficient Maltotriose utilization by industrial yeasts, but these new genes can be used in breeding and/or selection programs aimed at increasing yeast fitness for the efficient fermentation of brewer's wort.
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microarray karyotyping of maltose fermenting saccharomyces yeasts with differing Maltotriose utilization profiles reveals copy number variation in genes involved in maltose and Maltotriose utilization
Journal of Applied Microbiology, 2010Co-Authors: E H Duval, Boris U. Stambuk, Sérgio L. Alves, Barbara Dunn, Gavin SherlockAbstract: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.
Brian Gibson - One of the best experts on this subject based on the ideXlab platform.
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a deletion in the sta1 promoter determines Maltotriose and starch utilization in sta1 saccharomyces cerevisiae strains
Applied Microbiology and Biotechnology, 2019Co-Authors: Kristoffer Krogerus, Frederico Magalhães, Joosu Kuivanen, Brian GibsonAbstract:Diastatic strains of Saccharomyces cerevisiae are common contaminants in beer fermentations and are capable of producing an extracellular STA1-encoded glucoamylase. Recent studies have revealed variable diastatic ability in strains tested positive for STA1, and here, we elucidate genetic determinants behind this variation. We show that poorly diastatic strains have a 1162-bp deletion in the promoter of STA1. With CRISPR/Cas9-aided reverse engineering, we show that this deletion greatly decreases the ability to grow in beer and consume dextrin, and the expression of STA1. New PCR primers were designed for differentiation of highly and poorly diastatic strains based on the presence of the deletion in the STA1 promoter. In addition, using publically available whole genome sequence data, we show that the STA1 gene is prevalent among the ‘Beer 2’/‘Mosaic Beer’ brewing strains. These strains utilize Maltotriose efficiently, but the mechanisms for this have been unknown. By deleting STA1 from a number of highly diastatic strains, we show here that extracellular hydrolysis of Maltotriose through STA1 appears to be the dominant mechanism enabling Maltotriose use during wort fermentation in STA1+ strains. The formation and retention of STA1 seems to be an alternative evolutionary strategy for efficient utilization of sugars present in brewer’s wort. The results of this study allow for the improved reliability of molecular detection methods for diastatic contaminants in beer and can be exploited for strain development where Maltotriose use is desired.
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Evolutionary Engineering in Chemostat Cultures for Improved Maltotriose Fermentation Kinetics in Saccharomyces pastorianus Lager Brewing Yeast.
Frontiers in microbiology, 2017Co-Authors: Anja Brickwedde, Marcel Van Den Broek, Brian Gibson, Jack T. Pronk, Frederico Magalhães, Jan-maarten A. Geertman, Niels G. A. Kuijpers, Jean-marc DaranAbstract:The lager brewing yeast Saccharomyces pastorianus, an interspecies hybrid of S. eubayanus and S. cerevisiae, ferments Maltotriose, maltose, sucrose, glucose and fructose in wort to ethanol and carbon dioxide. Complete and timely conversion (‘attenuation’) of Maltotriose by industrial S. pastorianus strains is a key requirement for process intensification. This study explores a new evolutionary engineering strategy for improving Maltotriose fermentation kinetics. Prolonged carbon-limited, anaerobic chemostat cultivation of the reference strain S. pastorianus CBS1483 on a Maltotriose-enriched sugar mixture was used to select for spontaneous mutants with improved affinity for Maltotriose. Evolved populations exhibited an up to five-fold lower residual Maltotriose concentration and a higher ethanol concentration than the parental strain. Uptake studies with 14C-labelled sugars revealed an up to 4.75-fold higher transport capacity for Maltotriose in evolved strains. In laboratory batch cultures on wort, evolved strains showed improved attenuation and higher ethanol concentrations. These improvements were also observed in pilot fermentations at 1000-L scale with high-gravity wort. Although the evolved strain exhibited multiple chromosomal copy number changes, analysis of beer made from pilot fermentations showed no negative effects on flavour compound profiles. These results demonstrate the potential of evolutionary engineering for strain improvement of hybrid, alloploid brewing strains.
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Evolutionary Engineering in Chemostat Cultures for Improved Maltotriose Fermentation Kinetics in Saccharomyces pastorianus Lager Brewing Yeast
Frontiers Media S.A., 2017Co-Authors: Anja Brickwedde, Marcel Van Den Broek, Brian Gibson, Jack T. Pronk, Frederico Magalhães, Jan-maarten A. Geertman, Niels G. A. Kuijpers, Jean-marc DaranAbstract:The lager brewing yeast Saccharomyces pastorianus, an interspecies hybrid of S. eubayanus and S. cerevisiae, ferments Maltotriose, maltose, sucrose, glucose and fructose in wort to ethanol and carbon dioxide. Complete and timely conversion (“attenuation”) of Maltotriose by industrial S. pastorianus strains is a key requirement for process intensification. This study explores a new evolutionary engineering strategy for improving Maltotriose fermentation kinetics. Prolonged carbon-limited, anaerobic chemostat cultivation of the reference strain S. pastorianus CBS1483 on a Maltotriose-enriched sugar mixture was used to select for spontaneous mutants with improved affinity for Maltotriose. Evolved populations exhibited an up to 5-fold lower residual Maltotriose concentration and a higher ethanol concentration than the parental strain. Uptake studies with 14C-labeled sugars revealed an up to 4.75-fold higher transport capacity for Maltotriose in evolved strains. In laboratory batch cultures on wort, evolved strains showed improved attenuation and higher ethanol concentrations. These improvements were also observed in pilot fermentations at 1,000-L scale with high-gravity wort. Although the evolved strain exhibited multiple chromosomal copy number changes, analysis of beer made from pilot fermentations showed no negative effects on flavor compound profiles. These results demonstrate the potential of evolutionary engineering for strain improvement of hybrid, alloploid brewing strains
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Maltose and Maltotriose utilisation by group I strains of the hybrid lager yeast Saccharomyces pastorianus.
FEMS yeast research, 2016Co-Authors: Frederico Magalhães, Virve Vidgren, Laura Ruohonen, Brian GibsonAbstract:Brewer's wort is a challenging environment for yeast as it contains predominantly α-glucoside sugars. There exist two subgroups of the lager yeast Saccharomyces pastorianus which differ in sugar utilization. We performed wort fermentations and compared representative strains from both groups with respect to their ability to transport and ferment maltose and Maltotriose. Additionally, we mapped the transporters MALx1 , AGT1 , MPHx and MTT1 by Southern blotting. Contrary to previous observations, Group I comprises a diverse set of strains, with varying ability to transport and ferment Maltotriose. Of the eight Group I strains, three efficiently utilized Maltotriose, a property enabled by the presence of transmembrane transporters SeAGT1 and MTT1 . A58, a variant of the Group I type strain (CBS1513) performed particularly well, taking up Maltotriose at a higher rate than maltose and retaining significant transport activity at temperatures as low as 0°C. Analysis of transporter distribution in this strain revealed an increased copy number of the MTT1 gene, which encodes the only permease known with higher affinity for Maltotriose than maltose and low temperature dependence for transport. We propose that much of the variation in lager yeast fermentation behaviour is determined by the presence or absence of specific transmembrane transporters.
Jean-marc Daran - One of the best experts on this subject based on the ideXlab platform.
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Himalayan Saccharomyces eubayanus Genome Sequences Reveal Genetic Markers Explaining Heterotic Maltotriose Consumption by Saccharomyces pastorianus Hybrids.
Applied and environmental microbiology, 2019Co-Authors: Nick Brouwers, Arthur R. Gorter De Vries, Marcel Van Den Broek, Jack T. Pronk, Anja Brickwedde, Susan M Weening, Lieke Van Den Eijnden, Jasper A. Diderich, Feng-yan Bai, Jean-marc DaranAbstract:ABSTRACT Saccharomyces pastorianus strains are hybrids of Saccharomyces cerevisiae and Saccharomyces eubayanus that have been domesticated for centuries in lager beer brewing environments. As sequences and structures of S. pastorianus genomes are being resolved, molecular mechanisms and evolutionary origins of several industrially relevant phenotypes remain unknown. This study investigates how Maltotriose metabolism, a key feature in brewing, may have arisen in early S. eubayanus × S. cerevisiae hybrids. To address this question, we generated a nearly complete genome assembly of Himalayan S. eubayanus strains of the Holarctic subclade. This group of strains has been proposed to be the S. eubayanus subgenome origin of current S. pastorianus strains. The Himalayan S. eubayanus genomes harbored several copies of an S. eubayanusAGT1 (SeAGT1) α-oligoglucoside transporter gene with high sequence identity to genes encountered in S. pastorianus. Although Himalayan S. eubayanus strains cannot grow on maltose and Maltotriose, their maltose-hydrolase and SeMALT1 and SeAGT1 maltose transporter genes complemented the corresponding null mutants of S. cerevisiae. Expression, in Himalayan S. eubayanus of a functional S. cerevisiae maltose metabolism regulator gene (MALx3) enabled growth on oligoglucosides. The hypothesis that the Maltotriose-positive phenotype in S. pastorianus is a result of heterosis was experimentally tested by constructing an S. cerevisiae × S. eubayanus laboratory hybrid with a complement of maltose metabolism genes that resembles that of current S. pastorianus strains. The ability of this hybrid to consume Maltotriose in brewer’s wort demonstrated regulatory cross talk between subgenomes and thereby validated this hypothesis. These results support experimentally the new postulated hypothesis on the evolutionary origin of an essential phenotype of lager brewing strains and valuable knowledge for industrial exploitation of laboratory-made S. pastorianus-like hybrids. IMPORTANCES. pastorianus, an S. cerevisiae × S. eubayanus hybrid, is used for production of lager beer, the most produced alcoholic beverage worldwide. It emerged by spontaneous hybridization and colonized early lager brewing processes. Despite accumulation and analysis of genome sequencing data of S. pastorianus parental genomes, the genetic blueprint of industrially relevant phenotypes remains unresolved. Assimilation of Maltotriose, an abundant sugar in wort, has been postulated to be inherited from the S. cerevisiae parent. Here, we demonstrate that although Asian S. eubayanus isolates harbor a functional Maltotriose transporter SeAGT1 gene, they are unable to grow on α-oligoglucosides, but expression of S. cerevisiae regulator MAL13 (ScMAL13) was sufficient to restore growth on trisaccharides. We hypothesized that the S. pastorianus Maltotriose phenotype results from regulatory interaction between S. cerevisiae maltose transcription activator and the promoter of SeAGT1. We experimentally confirmed the heterotic nature of the phenotype, and thus these results provide experimental evidence of the evolutionary origin of an essential phenotype of lager brewing strains.
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Maltotriose consumption by hybrid saccharomyces pastorianus is heterotic and results from regulatory cross talk between parental sub genomes
bioRxiv, 2019Co-Authors: Nick Brouwers, Marcel Van Den Broek, Jack T. Pronk, Anja Brickwedde, Susan M Weening, Lieke Van Den Eijnden, Jasper A. Diderich, Feng-yan Bai, Arthur Gorter R. De Vries, Jean-marc DaranAbstract:S. pastorianus strains are hybrids of S. cerevisiae and S. eubayanus that have been domesticated for several centuries in lager-beer brewing environments. As sequences and structures of S. pastorianus genomes are being resolved, molecular mechanisms and evolutionary origin of several industrially relevant phenotypes remain unknown. This study investigates how Maltotriose metabolism, a key feature in brewing, may have arisen in early S. eubayanus x S. cerevisiae hybrids. To address this question, we generated a near-complete genome assembly of Himalayan S. eubayanus strains of the Holarctic subclade. This group of strains have been proposed to be the origin of the S. eubayanus subgenome of current S. pastorianus strains. The Himalayan S. eubayanus genomes harbored several copies of a SeAGT1 alpha-oligoglucoside transporter gene with high sequence identity to genes encountered in S. pastorianus. Although Himalayan S. eubayanus strains are unable to grown on maltose and Maltotriose, their maltose-hydrolase and SeMALT1 and SeAGT1 maltose-transporter genes complemented the corresponding null mutants of S. cerevisiae. Expression, in a Himalayan S. eubayanus strain, of a functional S. cerevisiae maltose-metabolism regulator gene (MALx3) enabled growth on oligoglucosides. The hypothesis that the Maltotriose-positive phenotype in S. pastorianus is a result of heterosis was experimentally tested by constructing a S. cerevisiae x S. eubayanus laboratory hybrid with a complement of maltose-metabolism genes that resembles that of current S. pastorianus strains. The ability of this hybrid to consume Maltotriose in brewer s wort demonstrated regulatory cross talk between sub-genomes and thereby validated this hypothesis. These results provide experimental evidence of the evolutionary origin of an essential phenotype of lager-brewing strains and valuable knowledge for industrial exploitation of laboratory-made S. pastorianus-like hybrids.
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In vivo recombination of Saccharomyces eubayanus maltose-transporter genes yields a chimeric transporter that enables Maltotriose fermentation
2019Co-Authors: Nick Brouwers, Marcel Van Den Broek, Jack T. Pronk, Niels G. A. Kuijpers, Susan M Weening, Arthur Gorter R. De Vries, Tom Elink D. Schuurman, Jean-marc DaranAbstract:Saccharomyces eubayanus is the non-S. cerevisiae parent of the lager-brewing hybrid S. pastorianus. In contrast to most S. cerevisiae and Frohberg-type S. pastorianus strains, S. eubayanus cannot utilize the α-tri-glucoside Maltotriose, a major carbohydrate in brewer’s wort. In Saccharomyces yeasts, utilization of Maltotriose is encoded by the subtelomeric MAL gene family, and requires transporters for Maltotriose uptake. While S. eubayanus strain CBS 12357T harbors four SeMALT genes which enable uptake of the α-di-glucoside maltose, it lacks Maltotriose transporter genes. In S. cerevisiae, sequence identity indicates that Maltotriose and maltose transporters likely evolved from a shared ancestral gene. To study the evolvability of Maltotriose utilization in S. eubayanus CBS 12357T, Maltotriose-assimilating mutants obtained after UV mutagenesis were subjected to laboratory evolution in carbon-limited chemostat cultures on Maltotriose-enriched wort. An evolved strain showed improved maltose and Maltotriose fermentation in 7 L fermenter experiments on industrial wort. Whole-genome sequencing revealed a novel mosaic SeMALT413 gene, resulting from repeated gene introgressions by non-reciprocal translocation of at least three SeMALT genes. The predicted tertiary structure of SeMalT413 was comparable to the original SeMalT transporters, but overexpression of SeMALT413 sufficed to enable growth on Maltotriose, indicating gene neofunctionalization had occurred. The mosaic structure of SeMALT413 resembles the structure of S. pastorianus Maltotriose-transporter gene SpMTY1, which has high sequences identity to alternatingly S. cerevisiae MALx1, S. paradoxus MALx1 and S. eubayanus SeMALT3. Evolution of the Maltotriose transporter landscape in hybrid S. pastorianus lager-brewing strains is therefore likely to have involved mechanisms similar to those observed in the present study.
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Mutagenesis and evolution to obtain Maltotriose consuming S. eubayanus.
2019Co-Authors: Nick Brouwers, Marcel Van Den Broek, Jack T. Pronk, Niels G. A. Kuijpers, Susan M Weening, Arthur Gorter R. De Vries, Tom Elink D. Schuurman, Jean-marc DaranAbstract:(A) Characterization of S. pastorianus CBS 1483(black), S. eubayanus CBS 12357T(blue) and IMS0637 (light red) on SMMt at 20°C. The data for IMS0637 is representative for the other mutants IMS0638-IMS0643 (S1 Fig). The average concentration of Maltotriose (diamonds) and average deviation were determined from two replicates (S2 Data File). (B) Characterization of S. pastorianus CBS 1483 (black), S. eubayanus CBS 12357T (blue) and IMS0637 (light red) on wort at 20°C. The concentrations of glucose (squares), maltose (triangles) and Maltotriose (diamonds) were measured from single biological measurements (S3 Data File). (C) Residual Maltotriose concentration in the outflow during laboratory evolution of strains IMS0637-IMS0643 in an anaerobic chemostat at 20°C on Maltotriose enriched wort. The concentrations of glucose (squares), maltose (triangles) and Maltotriose (diamonds) were measured by HPLC. The chemostat was restarted after a technical failure (red dotted line, S4 Data File). (D) Characterization of S. pastorianus CBS 1483 (black), S. eubayanus CBS 12357T (blue) and IMS0750 (red) on wort at 12°C in 250 mL micro-aerobic Neubor infusion bottles. The average concentration and standard deviation of glucose (squares), maltose (triangles) and Maltotriose (diamonds) were determined from three biological replicates. The data for IMS0751 and IMS0752 are shown in S5 Data File and S2 Fig.
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Organization of maltose and Maltotriose transporter genes in S. cerevisiae and S. eubayanus.
2019Co-Authors: Nick Brouwers, Marcel Van Den Broek, Jack T. Pronk, Niels G. A. Kuijpers, Susan M Weening, Arthur Gorter R. De Vries, Tom Elink D. Schuurman, Jean-marc DaranAbstract:In Saccharomyces species, maltose and Maltotriose utilization is encoded in the MAL genes, which are located in subtelomeric regions and comprise three types of genes: a MALT α-oligo-glucoside proton-symporter gene, a MALS α-glucosidase gene which hydrolyses α-(di or tri)-glucosides into glucose, and a MALR regulator gene that induces the transcription of MALT and MALS genes in the presence of maltose. In canonical MAL loci, the MALT and MALS are expressed from a bi-directional MALR-dependent promoter sequence. The chromosomal location of known maltose and Maltotriose transporter genes in S. cerevisiae and S. eubayanus is shown, although the presence of these genes varies among isolates. ScMPH2 and ScMPH3 encode α-glucoside permeases which do not enable efficient Maltotriose uptake [11]. ScMAL31, ScMAL21, ScMAL61 and ScMAL41 encode maltose transporters of the ScMalx1 family. ScAGT1 encodes a Maltotriose transporter. SeMALT1, SeMALT2, SeMALT3 and SeMALT4 encode maltose transporters with high sequence identity to the ScMalx1 family. SeAGT1 is an Maltotriose transporter which has recently been discovered in the north American S. eubayanus isolate yHRVM108 [19].
Kristoffer Krogerus - One of the best experts on this subject based on the ideXlab platform.
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a deletion in the sta1 promoter determines Maltotriose and starch utilization in sta1 saccharomyces cerevisiae strains
Applied Microbiology and Biotechnology, 2019Co-Authors: Kristoffer Krogerus, Frederico Magalhães, Joosu Kuivanen, Brian GibsonAbstract:Diastatic strains of Saccharomyces cerevisiae are common contaminants in beer fermentations and are capable of producing an extracellular STA1-encoded glucoamylase. Recent studies have revealed variable diastatic ability in strains tested positive for STA1, and here, we elucidate genetic determinants behind this variation. We show that poorly diastatic strains have a 1162-bp deletion in the promoter of STA1. With CRISPR/Cas9-aided reverse engineering, we show that this deletion greatly decreases the ability to grow in beer and consume dextrin, and the expression of STA1. New PCR primers were designed for differentiation of highly and poorly diastatic strains based on the presence of the deletion in the STA1 promoter. In addition, using publically available whole genome sequence data, we show that the STA1 gene is prevalent among the ‘Beer 2’/‘Mosaic Beer’ brewing strains. These strains utilize Maltotriose efficiently, but the mechanisms for this have been unknown. By deleting STA1 from a number of highly diastatic strains, we show here that extracellular hydrolysis of Maltotriose through STA1 appears to be the dominant mechanism enabling Maltotriose use during wort fermentation in STA1+ strains. The formation and retention of STA1 seems to be an alternative evolutionary strategy for efficient utilization of sugars present in brewer’s wort. The results of this study allow for the improved reliability of molecular detection methods for diastatic contaminants in beer and can be exploited for strain development where Maltotriose use is desired.
Frederico Magalhães - One of the best experts on this subject based on the ideXlab platform.
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a deletion in the sta1 promoter determines Maltotriose and starch utilization in sta1 saccharomyces cerevisiae strains
Applied Microbiology and Biotechnology, 2019Co-Authors: Kristoffer Krogerus, Frederico Magalhães, Joosu Kuivanen, Brian GibsonAbstract:Diastatic strains of Saccharomyces cerevisiae are common contaminants in beer fermentations and are capable of producing an extracellular STA1-encoded glucoamylase. Recent studies have revealed variable diastatic ability in strains tested positive for STA1, and here, we elucidate genetic determinants behind this variation. We show that poorly diastatic strains have a 1162-bp deletion in the promoter of STA1. With CRISPR/Cas9-aided reverse engineering, we show that this deletion greatly decreases the ability to grow in beer and consume dextrin, and the expression of STA1. New PCR primers were designed for differentiation of highly and poorly diastatic strains based on the presence of the deletion in the STA1 promoter. In addition, using publically available whole genome sequence data, we show that the STA1 gene is prevalent among the ‘Beer 2’/‘Mosaic Beer’ brewing strains. These strains utilize Maltotriose efficiently, but the mechanisms for this have been unknown. By deleting STA1 from a number of highly diastatic strains, we show here that extracellular hydrolysis of Maltotriose through STA1 appears to be the dominant mechanism enabling Maltotriose use during wort fermentation in STA1+ strains. The formation and retention of STA1 seems to be an alternative evolutionary strategy for efficient utilization of sugars present in brewer’s wort. The results of this study allow for the improved reliability of molecular detection methods for diastatic contaminants in beer and can be exploited for strain development where Maltotriose use is desired.
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Evolutionary Engineering in Chemostat Cultures for Improved Maltotriose Fermentation Kinetics in Saccharomyces pastorianus Lager Brewing Yeast.
Frontiers in microbiology, 2017Co-Authors: Anja Brickwedde, Marcel Van Den Broek, Brian Gibson, Jack T. Pronk, Frederico Magalhães, Jan-maarten A. Geertman, Niels G. A. Kuijpers, Jean-marc DaranAbstract:The lager brewing yeast Saccharomyces pastorianus, an interspecies hybrid of S. eubayanus and S. cerevisiae, ferments Maltotriose, maltose, sucrose, glucose and fructose in wort to ethanol and carbon dioxide. Complete and timely conversion (‘attenuation’) of Maltotriose by industrial S. pastorianus strains is a key requirement for process intensification. This study explores a new evolutionary engineering strategy for improving Maltotriose fermentation kinetics. Prolonged carbon-limited, anaerobic chemostat cultivation of the reference strain S. pastorianus CBS1483 on a Maltotriose-enriched sugar mixture was used to select for spontaneous mutants with improved affinity for Maltotriose. Evolved populations exhibited an up to five-fold lower residual Maltotriose concentration and a higher ethanol concentration than the parental strain. Uptake studies with 14C-labelled sugars revealed an up to 4.75-fold higher transport capacity for Maltotriose in evolved strains. In laboratory batch cultures on wort, evolved strains showed improved attenuation and higher ethanol concentrations. These improvements were also observed in pilot fermentations at 1000-L scale with high-gravity wort. Although the evolved strain exhibited multiple chromosomal copy number changes, analysis of beer made from pilot fermentations showed no negative effects on flavour compound profiles. These results demonstrate the potential of evolutionary engineering for strain improvement of hybrid, alloploid brewing strains.
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Evolutionary Engineering in Chemostat Cultures for Improved Maltotriose Fermentation Kinetics in Saccharomyces pastorianus Lager Brewing Yeast
Frontiers Media S.A., 2017Co-Authors: Anja Brickwedde, Marcel Van Den Broek, Brian Gibson, Jack T. Pronk, Frederico Magalhães, Jan-maarten A. Geertman, Niels G. A. Kuijpers, Jean-marc DaranAbstract:The lager brewing yeast Saccharomyces pastorianus, an interspecies hybrid of S. eubayanus and S. cerevisiae, ferments Maltotriose, maltose, sucrose, glucose and fructose in wort to ethanol and carbon dioxide. Complete and timely conversion (“attenuation”) of Maltotriose by industrial S. pastorianus strains is a key requirement for process intensification. This study explores a new evolutionary engineering strategy for improving Maltotriose fermentation kinetics. Prolonged carbon-limited, anaerobic chemostat cultivation of the reference strain S. pastorianus CBS1483 on a Maltotriose-enriched sugar mixture was used to select for spontaneous mutants with improved affinity for Maltotriose. Evolved populations exhibited an up to 5-fold lower residual Maltotriose concentration and a higher ethanol concentration than the parental strain. Uptake studies with 14C-labeled sugars revealed an up to 4.75-fold higher transport capacity for Maltotriose in evolved strains. In laboratory batch cultures on wort, evolved strains showed improved attenuation and higher ethanol concentrations. These improvements were also observed in pilot fermentations at 1,000-L scale with high-gravity wort. Although the evolved strain exhibited multiple chromosomal copy number changes, analysis of beer made from pilot fermentations showed no negative effects on flavor compound profiles. These results demonstrate the potential of evolutionary engineering for strain improvement of hybrid, alloploid brewing strains
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Maltose and Maltotriose utilisation by group I strains of the hybrid lager yeast Saccharomyces pastorianus.
FEMS yeast research, 2016Co-Authors: Frederico Magalhães, Virve Vidgren, Laura Ruohonen, Brian GibsonAbstract:Brewer's wort is a challenging environment for yeast as it contains predominantly α-glucoside sugars. There exist two subgroups of the lager yeast Saccharomyces pastorianus which differ in sugar utilization. We performed wort fermentations and compared representative strains from both groups with respect to their ability to transport and ferment maltose and Maltotriose. Additionally, we mapped the transporters MALx1 , AGT1 , MPHx and MTT1 by Southern blotting. Contrary to previous observations, Group I comprises a diverse set of strains, with varying ability to transport and ferment Maltotriose. Of the eight Group I strains, three efficiently utilized Maltotriose, a property enabled by the presence of transmembrane transporters SeAGT1 and MTT1 . A58, a variant of the Group I type strain (CBS1513) performed particularly well, taking up Maltotriose at a higher rate than maltose and retaining significant transport activity at temperatures as low as 0°C. Analysis of transporter distribution in this strain revealed an increased copy number of the MTT1 gene, which encodes the only permease known with higher affinity for Maltotriose than maltose and low temperature dependence for transport. We propose that much of the variation in lager yeast fermentation behaviour is determined by the presence or absence of specific transmembrane transporters.