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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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CRISPR-Cas9 mediated gene deletions in lager yeast Saccharomyces pastorianus.
Microbial cell factories, 2017Co-Authors: Arthur R. Gorter De Vries, Philip A. De Groot, Marcel Van Den Broek, Jean-marc DaranAbstract:Background The ease of use of CRISPR-Cas9 reprogramming, its high efficacy, and its multiplexing capabilities have brought this technology at the forefront of genome editing techniques. Saccharomyces pastorianus is an aneuploid interspecific hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus that has been domesticated for centuries and is used for the industrial fermentation of lager beer. For yet uncharacterised reasons, this hybrid yeast is far more resilient to genetic alteration than its ancestor S. cerevisiae.
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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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functional analysis and transcriptional regulation of two orthologs of aro10 encoding broad substrate specificity 2 oxo acid decarboxylases in the brewing yeast Saccharomyces pastorianus cbs1483
Fems Yeast Research, 2013Co-Authors: Irina Bolat, Jack T. Pronk, Gabriele Romagnoli, Feibai Zhu, Jean-marc DaranAbstract:The hybrid genomes of Saccharomyces pastorianus consist of subgenomes similar to those of S. cerevisiae and S. eubayanus, and impact of the genome structure on flavour production and its regulation is poorly understood. This study focuses on ARO10 , a 2-oxo-acid decarboxylase involved in production of higher alcohols. In S. pastorianus CBS1483, four ARO10 copies were identified, three resembled S. cerevisiae ARO10 and one S. eubayanus ARO10 . Substrate specificities of lager strain (Lg)ScAro10 and LgSeubAro10 were compared by individually expressing them in a pdc1Δ-pdc5Δ-pdc6Δ-aro10Δ-thi3Δ S. cerevisiae strain. Both isoenzymes catalysed decarboxylation of the 2-oxo-acids derived from branched-chain, sulphur-containing amino acids and preferably phenylpyruvate. Expression of both alleles was induced by phenylalanine, however in contrast to the S. cerevisiae strain, the two genes were not induced by leucine. Additionally, LgSeubARO10 showed higher basal expression levels during growth with ammonia. ARO80 , which encodes ARO10 transcriptional activator, is located on CHRIV and counts three Sc- like and one Seub -like copies. Deletion of LgSeubARO80 did not affect LgSeubARO10 phenylalanine induction, revealing ‘trans’ regulation across the subgenomes. ARO10 transcript levels showed a poor correlation with decarboxylase activities. These results provide insights into flavour formation in S. pastorianus and illustrate the complexity of functional characterization in aneuploid strains.
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Functional analysis and transcriptional regulation of two orthologs of ARO10, encoding broad‐substrate‐specificity 2‐oxo‐acid decarboxylases, in the brewing yeast Saccharomyces pastorianus CBS1483
FEMS yeast research, 2013Co-Authors: Irina Bolat, Jack T. Pronk, Gabriele Romagnoli, Feibai Zhu, Jean-marc DaranAbstract:The hybrid genomes of Saccharomyces pastorianus consist of subgenomes similar to those of S. cerevisiae and S. eubayanus, and impact of the genome structure on flavour production and its regulation is poorly understood. This study focuses on ARO10 , a 2-oxo-acid decarboxylase involved in production of higher alcohols. In S. pastorianus CBS1483, four ARO10 copies were identified, three resembled S. cerevisiae ARO10 and one S. eubayanus ARO10 . Substrate specificities of lager strain (Lg)ScAro10 and LgSeubAro10 were compared by individually expressing them in a pdc1Δ-pdc5Δ-pdc6Δ-aro10Δ-thi3Δ S. cerevisiae strain. Both isoenzymes catalysed decarboxylation of the 2-oxo-acids derived from branched-chain, sulphur-containing amino acids and preferably phenylpyruvate. Expression of both alleles was induced by phenylalanine, however in contrast to the S. cerevisiae strain, the two genes were not induced by leucine. Additionally, LgSeubARO10 showed higher basal expression levels during growth with ammonia. ARO80 , which encodes ARO10 transcriptional activator, is located on CHRIV and counts three Sc- like and one Seub -like copies. Deletion of LgSeubARO80 did not affect LgSeubARO10 phenylalanine induction, revealing ‘trans’ regulation across the subgenomes. ARO10 transcript levels showed a poor correlation with decarboxylase activities. These results provide insights into flavour formation in S. pastorianus and illustrate the complexity of functional characterization in aneuploid strains.
T Becker - One of the best experts on this subject based on the ideXlab platform.
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induced expression of the alcohol acetyltransferase gene atf1 in industrial yeast Saccharomyces pastorianus tum 34 70
Yeast, 2018Co-Authors: S Fischer, K R Buchner, T BeckerAbstract:Targeted induced gene expression for industrial fermentation processes in food and beverage production could fulfill future demands. To avoid metabolic burden and disturbances owing to the fermentation procedure, induced gene expression is necessary for combating stress, such as that caused by temperature shifts that occur during the transition from fermentation to maturation in the brewing process. The aim of this study was to target gene expression in industrial yeast using stress-responsive promoters and homologues of the selection marker SMR1. Self-cloning strains of the industrial brewing yeast Saccharomyces pastorianus TUM 34/70 were constructed to overexpress the alcohol acetyltransferase (ATF1) gene under the control of inducible promoters P SSA3, P HSP104 and P UBI4. Transcription analysis shows the highest induction after 72 h of shock situation for P HSP104 with 1.3-fold and P UBI4 with 2.2-fold. Further, at the end of shock situation the concentrations of ethyl acetate were 1.2- and 1.3-fold higher than the wild type for P HSP104 and P UBI4, respectively. In addition, the influence of the final temperature and temporal sequence of temperature shock to 4°C had a major impact on expression patterns. Therefore, these data show that temperature-induced gene expression of self-cloning industrial yeast could be an option for optimization of the beverage fermentation.
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Induced expression of the alcohol acetyltransferase gene ATF1 in industrial yeast Saccharomyces pastorianus TUM 34/70.
Yeast (Chichester England), 2018Co-Authors: S Fischer, K R Buchner, T BeckerAbstract:Targeted induced gene expression for industrial fermentation processes in food and beverage production could fulfill future demands. To avoid metabolic burden and disturbances owing to the fermentation procedure, induced gene expression is necessary for combating stress, such as that caused by temperature shifts that occur during the transition from fermentation to maturation in the brewing process. The aim of this study was to target gene expression in industrial yeast using stress-responsive promoters and homologues of the selection marker SMR1. Self-cloning strains of the industrial brewing yeast Saccharomyces pastorianus TUM 34/70 were constructed to overexpress the alcohol acetyltransferase (ATF1) gene under the control of inducible promoters P SSA3, P HSP104 and P UBI4. Transcription analysis shows the highest induction after 72 h of shock situation for P HSP104 with 1.3-fold and P UBI4 with 2.2-fold. Further, at the end of shock situation the concentrations of ethyl acetate were 1.2- and 1.3-fold higher than the wild type for P HSP104 and P UBI4, respectively. In addition, the influence of the final temperature and temporal sequence of temperature shock to 4°C had a major impact on expression patterns. Therefore, these data show that temperature-induced gene expression of self-cloning industrial yeast could be an option for optimization of the beverage fermentation.
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induced gene expression in industrial Saccharomyces pastorianus var carlsbergensis tum 34 70 evaluation of temperature and ethanol inducible native promoters
Fems Yeast Research, 2016Co-Authors: S Fischer, C. Engstler, Susanne Procopio, T BeckerAbstract:Induced gene expression is an important trait in yeast metabolic engineering, but current regulations prevent the use of conventional expression systems, such as galactose and copper, in food and beverage fermentations. This article examines the suitability of temperature-inducible native promoters for use in the industrial yeast strain Saccharomyces pastorianus var. carlsbergensis TUM 34/70 under brewing conditions . Ten different promoters were cloned and characterized under varying temperature shifts and ethanol concentrations using a green fluorescent protein reporter. The activities of these promoters varied depending upon the stress conditions applied. A temperature shift to 4°C led to the highest fold changes of PSSA3, PUBI4 and PHSP104 by 5.4, 4.5 and 5.0, respectively. Ethanol shock at 24°C showed marked, concentration-dependent induction of the promoters. Here, PHSP104 showed its highest induction at ethanol concentrations between 4% (v/v) and 6% (v/v). The highest fold changes of PSSA3 and PUBI4 were found at 10% (v/v) ethanol. In comparison, the ethanol shock at a typical fermentation temperature (12°C) leads to lower induction patterns of these promoters. Taken together, the data show that three promoters (PHSP104, PUBI4 and PSSA3) have high potential for targeted gene expression in self-cloning brewing yeast using temperature shifts.
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Induced gene expression in industrial Saccharomyces pastorianus var. carlsbergensis TUM 34/70: evaluation of temperature and ethanol inducible native promoters.
FEMS yeast research, 2016Co-Authors: S Fischer, C. Engstler, Susanne Procopio, T BeckerAbstract:Induced gene expression is an important trait in yeast metabolic engineering, but current regulations prevent the use of conventional expression systems, such as galactose and copper, in food and beverage fermentations. This article examines the suitability of temperature-inducible native promoters for use in the industrial yeast strain Saccharomyces pastorianus var. carlsbergensis TUM 34/70 under brewing conditions . Ten different promoters were cloned and characterized under varying temperature shifts and ethanol concentrations using a green fluorescent protein reporter. The activities of these promoters varied depending upon the stress conditions applied. A temperature shift to 4°C led to the highest fold changes of PSSA3, PUBI4 and PHSP104 by 5.4, 4.5 and 5.0, respectively. Ethanol shock at 24°C showed marked, concentration-dependent induction of the promoters. Here, PHSP104 showed its highest induction at ethanol concentrations between 4% (v/v) and 6% (v/v). The highest fold changes of PSSA3 and PUBI4 were found at 10% (v/v) ethanol. In comparison, the ethanol shock at a typical fermentation temperature (12°C) leads to lower induction patterns of these promoters. Taken together, the data show that three promoters (PHSP104, PUBI4 and PSSA3) have high potential for targeted gene expression in self-cloning brewing yeast using temperature shifts.
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Part II: the influence of the serial repitching of Saccharomyces pastorianus on the uptake dynamics of amino acids during the fermentation of barley and gluten‐free buckwheat and quinoa wort
Journal of the Institute of Brewing, 2015Co-Authors: Matjaž Deželak, T Becker, Mekonnen M. Gebremariam, Martin Zarnkow, Iztok Jože KoširAbstract:The present paper is part of a comprehensive study regarding the influence of the serial repitching of Saccharomyces pastorianus TUM 34/70 on the composition of the barley, buckwheat and quinoa fermentation medium. In particular, it focuses on the uptake dynamics of amino acids during 11 successive fermentations. Samples were taken every 20 h after pitching, analysed for the particular amino acid content and statistically evaluated. The term ‘completion time’ (t95), here defined as the percentage attenuation time necessary for ~95% of the total assimilation, has been introduced. In addition, ‘the serial repitching factor’ is used for the first time to support the visual evaluation of the influence of serial repitching. Amino acids that were essentially affected by serial repitching were glutamine, arginine, alanine and tryptophan in barley, aspartate, glutamate and tryptophan in buckwheat, and all in the quinoa wort fermentation. As opposed to buckwheat and quinoa, in barley the amino acids behaved more or less independently from each other, which for buckwheat and quinoa indicates a more general systemic change in the yeast. From the amino acids point of view, buckwheat can be fully regarded as a suitable gluten-free substitute for barley beer since the amino acid assimilation was very consistent and hardly influenced by the serial repitching, especially regarding the final amino acid assimilation. In the case of quinoa, the assimilation of all amino acids became significantly affected after the sixth fermentation and quinoa is probably unsuitable for the production of beer-like beverages. Results suggest no substitutional potential of quinoa for barley beer, but if a nutrient-rich beverage of choice from quinoa malt is intended to be prepared, it seems that the serial repitching is limited to six fermentations at most. Copyright © 2015 The Institute of Brewing & Distilling
Jack T. Pronk - One of the best experts on this subject based on the ideXlab platform.
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Improving Industrially Relevant Phenotypic Traits by Engineering Chromosome Copy Number in Saccharomyces pastorianus.
Frontiers in genetics, 2020Co-Authors: Arthur R. Gorter De Vries, Marcel Van Den Broek, Ewout Knibbe, Roderick Van Roosmalen, Pilar De La Torre Cortés, Stephanie F O'herne, Pascal A Vijverberg, Anissa El Masoudi, Nick Brouwers, Jack T. PronkAbstract:The lager-brewing yeast Saccharomyces pastorianus is a hybrid between S. cerevisiae and S. eubayanus with an exceptional degree of aneuploidy. While chromosome copy number variation (CCNV) is present in many industrial Saccharomyces strains and has been linked to various industrially-relevant traits, its impact on the brewing performance of S. pastorianus remains elusive. Here we attempt to delete single copies of chromosomes which are relevant for the production of off-flavor compound diacetyl by centromere silencing. However, the engineered strains display CNV of multiple non-targeted chromosomes. We attribute this unintended CCNV to inherent instability and to a mutagenic effect of electroporation and of centromere-silencing. Regardless, the resulting strains displayed large phenotypic diversity. By growing centromere-silenced cells in repeated sequential batches in medium containing 10% ethanol, mutants with increased ethanol tolerance were obtained. By using CCNV mutagenesis by exposure to the mitotic inhibitor MBC, selection in the same set-up yielded even more tolerant mutants that would not classify as genetically modified organisms. These results show that CCNV of alloaneuploid S. pastorianus genomes is highly unstable, and that CCNV mutagenesis can generate broad diversity. Coupled to effective selection or screening, CCNV mutagenesis presents a potent tool for strain improvement.
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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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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 G. 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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Chromosomal Copy Number Variation in Saccharomyces pastorianus Is Evidence for Extensive Genome Dynamics in Industrial Lager Brewing Strains
Applied and environmental microbiology, 2015Co-Authors: M. Van Den Broek, Jack T. Pronk, Irina Bolat, Jurgen F. Nijkamp, Edward Ramos, Marijke A. H. Luttik, Frank Koopman, J. M. Geertman, Denise T. D. De Ridder, J.m. DaranAbstract: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.
Irina Bolat - One of the best experts on this subject based on the ideXlab platform.
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Chromosomal Copy Number Variation in Saccharomyces pastorianus Is Evidence for Extensive Genome Dynamics in Industrial Lager Brewing Strains
Applied and environmental microbiology, 2015Co-Authors: M. Van Den Broek, Jack T. Pronk, Irina Bolat, Jurgen F. Nijkamp, Edward Ramos, Marijke A. H. Luttik, Frank Koopman, J. M. Geertman, Denise T. D. De Ridder, J.m. DaranAbstract: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.
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functional analysis and transcriptional regulation of two orthologs of aro10 encoding broad substrate specificity 2 oxo acid decarboxylases in the brewing yeast Saccharomyces pastorianus cbs1483
Fems Yeast Research, 2013Co-Authors: Irina Bolat, Jack T. Pronk, Gabriele Romagnoli, Feibai Zhu, Jean-marc DaranAbstract:The hybrid genomes of Saccharomyces pastorianus consist of subgenomes similar to those of S. cerevisiae and S. eubayanus, and impact of the genome structure on flavour production and its regulation is poorly understood. This study focuses on ARO10 , a 2-oxo-acid decarboxylase involved in production of higher alcohols. In S. pastorianus CBS1483, four ARO10 copies were identified, three resembled S. cerevisiae ARO10 and one S. eubayanus ARO10 . Substrate specificities of lager strain (Lg)ScAro10 and LgSeubAro10 were compared by individually expressing them in a pdc1Δ-pdc5Δ-pdc6Δ-aro10Δ-thi3Δ S. cerevisiae strain. Both isoenzymes catalysed decarboxylation of the 2-oxo-acids derived from branched-chain, sulphur-containing amino acids and preferably phenylpyruvate. Expression of both alleles was induced by phenylalanine, however in contrast to the S. cerevisiae strain, the two genes were not induced by leucine. Additionally, LgSeubARO10 showed higher basal expression levels during growth with ammonia. ARO80 , which encodes ARO10 transcriptional activator, is located on CHRIV and counts three Sc- like and one Seub -like copies. Deletion of LgSeubARO80 did not affect LgSeubARO10 phenylalanine induction, revealing ‘trans’ regulation across the subgenomes. ARO10 transcript levels showed a poor correlation with decarboxylase activities. These results provide insights into flavour formation in S. pastorianus and illustrate the complexity of functional characterization in aneuploid strains.
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Functional analysis and transcriptional regulation of two orthologs of ARO10, encoding broad‐substrate‐specificity 2‐oxo‐acid decarboxylases, in the brewing yeast Saccharomyces pastorianus CBS1483
FEMS yeast research, 2013Co-Authors: Irina Bolat, Jack T. Pronk, Gabriele Romagnoli, Feibai Zhu, Jean-marc DaranAbstract:The hybrid genomes of Saccharomyces pastorianus consist of subgenomes similar to those of S. cerevisiae and S. eubayanus, and impact of the genome structure on flavour production and its regulation is poorly understood. This study focuses on ARO10 , a 2-oxo-acid decarboxylase involved in production of higher alcohols. In S. pastorianus CBS1483, four ARO10 copies were identified, three resembled S. cerevisiae ARO10 and one S. eubayanus ARO10 . Substrate specificities of lager strain (Lg)ScAro10 and LgSeubAro10 were compared by individually expressing them in a pdc1Δ-pdc5Δ-pdc6Δ-aro10Δ-thi3Δ S. cerevisiae strain. Both isoenzymes catalysed decarboxylation of the 2-oxo-acids derived from branched-chain, sulphur-containing amino acids and preferably phenylpyruvate. Expression of both alleles was induced by phenylalanine, however in contrast to the S. cerevisiae strain, the two genes were not induced by leucine. Additionally, LgSeubARO10 showed higher basal expression levels during growth with ammonia. ARO80 , which encodes ARO10 transcriptional activator, is located on CHRIV and counts three Sc- like and one Seub -like copies. Deletion of LgSeubARO80 did not affect LgSeubARO10 phenylalanine induction, revealing ‘trans’ regulation across the subgenomes. ARO10 transcript levels showed a poor correlation with decarboxylase activities. These results provide insights into flavour formation in S. pastorianus and illustrate the complexity of functional characterization in aneuploid strains.
Sachiko Odake - One of the best experts on this subject based on the ideXlab platform.
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Temperature-dependency on the inactivation of Saccharomyces pastorianus by low-pressure carbon dioxide microbubbles.
Journal of food science and technology, 2019Co-Authors: Fumiyuki Kobayashi, Sachiko OdakeAbstract:Temperature-dependency on cell membrane injury and inactivation of Saccharomyces pastorianus by low-pressure carbon dioxide microbubbles (MBCO2) was investigated. The number of surviving S. pastorianus cells after MBCO2 treatment detected with yeast and mould agar (YMA, an optimum agar) was higher than that with YMA adding 2.5 g/L sodium chloride and yeast nitrogen base agar (a minimum agar). However, the decrease of the surviving number by thermal treatment was not changed among above agars used. The fluorescence polarization (FP), which indicated the phase transition of the membrane of S. pastorianus cells treated with MBCO2 increased with increasing temperature. The activity of the alkaline phosphatase (AP), a periplasmic enzyme, in S. pastorianus cells after MBCO2 and thermal treatments increased with the FP but was reduced by further increasing temperature. The FP and AP activities after MBCO2 treatment increased at a temperature lower than the temperature of the thermal treatment. In addition, intracellular pH of S. pastorianus decreased by the MBCO2 treatment at lower temperature with increasing pressure. Therefore, it was revealed that phase transition of the cell membrane and inactivation of S. pastorianus was caused by MBCO2 treatment at lower temperature than thermal treatment and that the effect was induced by the dissolved CO2 and increased with increasing pressure.
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The relationship between intracellular acidification and inactivation of Saccharomyces pastorianus by a two-stage system with pressurized carbon dioxide microbubbles
Biochemical Engineering Journal, 2018Co-Authors: Fumiyuki Kobayashi, Sachiko OdakeAbstract:Abstract To investigate the relationship between intracellular acidification and inactivation of Saccharomyces pastorianus by a two-stage system with pressurized carbon dioxide microbubbles (two-stage MBCO2), the survivors and intracellular pH (pHin) of S. pastorianus suspended in buffers at various pHs after the two-stage MBCO2 treatment were measured. The inactivation efficiency of two-stage MBCO2 on S. pastorianus increased with lowering the initial buffer pH, although the pHin of S. pastorianus in pH 3 buffer was decreased by heating to 45 °C, regardless of the use of MBCO2. Conversely, the number of surviving S. pastorianus cells in pH 4 and 5 buffers was decreased by the two-stage MBCO2 without the pHin-lowering. Furthermore, the inactivation efficiency of two-stage MBCO2 on S. pastorianus increased with increasing pressure in the mixing vessel, although the pHin-lowering was caused by heating to 45 °C for all of the pressures tested, regardless of the use of MBCO2. Therefore, intracellular acidification of S. pastorianus by two-stage MBCO2 was due to the cell penetration of extracellular H+ associated with the temperature increase, and the inactivation was suggested to be induced by the combined factors of increasing temperature, pHin- and pHex-lowering and high dissolved CO2 concentration.
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Ethanol addition on inactivation of Saccharomyces pastorianus by a two-stage system with low-pressure carbon dioxide microbubbles can accelerate the cell membrane injury.
Biotechnology progress, 2017Co-Authors: Fumiyuki Kobayashi, Sachiko OdakeAbstract:The effect of ethanol on the inactivation of Saccharomyces pastorianus by a two-stage system with low-pressure carbon dioxide microbubbles (two-stage MBCO2 ) was investigated. Zero and >5 log reductions of S. pastorianus populations suspended in physiological saline (PS) containing 0% and 10% ethanol, respectively, occurred by the two-stage MBCO2 at a mixing vessel pressure of 1 MPa and a heating coil temperature of 40°C. Conversely, the detected number of surviving S. pastorianus cells in PS containing 5% ethanol was higher in yeast and mold agar (YMA, an optimum agar) than YMA with 2.5% sodium chloride, followed by yeast nitrogen base agar (YNBA, a minimum agar). The fluorescence polarization of S. pastorianus in PS containing 5% and 10% ethanol increased similarly with exposure time in the heating coil of two-stage MBCO2 and was correlated with the surviving cell number measured in YNBA. The intracellular pH (pHin ) of S. pastorianus in PS containing 5% ethanol decreased linearly with exposure time in the heating coil of two-stage MBCO2 . Also, the pHin -lowering of S. pastorianus in PS containing 10% ethanol was drastically caused by two-stage MBCO2 at 1 min exposure time in the heating coil but then stayed constant until 5 min, agreeing with the inactivation efficiency. Therefore, ethanol in S. pastorianus suspension was suggested to accelerate the cell membrane injury caused by two-stage MBCO2 . © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:282-286, 2018.
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Intracellular acidification and damage of cellular membrane of Saccharomyces pastorianus by low-pressure carbon dioxide microbubbles
Food Control, 2017Co-Authors: Fumiyuki Kobayashi, Sachiko OdakeAbstract:Abstract The effects of intracellular acidification and damage of cellular membrane on the inactivation of Saccharomyces pastorianus by a two-stage method of low-pressure carbon dioxide microbubbles (two-stage MBCO2) were investigated. Intra/extra cellular pH (pHin and pHex) and propidium iodide (PI) uptake of S. pastorianus cells treated with the two-stage MBCO2 were measured by fluorescence analysis, and were compared with the inactivation efficiency. The pHex and pHin of S. pastorianus cells treated with only mixing vessel at 10 °C of two-stage MBCO2 was lower than those that were untreated. However, the surviving number and the pHin of S. pastorianus cells treated with the two-stage MBCO2 containing the mixing vessel at 1.0 MPa and the heating coil of 40 °C decreased concomitant with the exposure time, and the increase of the PI uptake ratio was confirmed. In addition, when the pressure of the mixing vessel was 1.0 MPa, the surviving number and the pHin of S. pastorianus cells significantly decreased, and the PI uptake ratio increased by increasing the temperature to 45 and 50 °C with the heating coil of the two-stage MBCO2. These results suggested that the inactivation of S. pastorianus by two-stage MBCO2 might be induced by the damage to the cellular membrane at 45 and 50 °C, and by the lowering of the pHin at 40 °C.
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comparison of a two stage system with low pressure carbon dioxide microbubbles and heat treatment on the inactivation of Saccharomyces pastorianus cells
Food Control, 2014Co-Authors: Fumiyuki Kobayashi, Sachiko Odake, Masaki Sugiura, Hiromi Ikeura, Michio Sato, Masahiko TamakiAbstract:To clarify how Saccharomyces pastorianus cells were affected by a two-stage system that was heating and pressurizing after microbubbled carbon dioxide (MB-CO2) was mixed with the S. pastorianus suspension at low temperature and pressure (two-stage MB-CO2), S. pastorianus cells were observed by electronic microscopy and stained with propidium iodide (PI). Furthermore, the amounts of nucleic acid and protein leaked from treated S. pastorianus cells were determined and intracellular enzyme activities were measured. It was observed by scanning electric microscopy that wrinkles in S. pastorianus cells treated by two-stage MB-CO2 with a heating coil at 50 °C (MB50) and heat treatments at 50 °C and 80 °C (H50 and H80) were more than those in untreated (NT) cells. Upon observation with transmission electron microscopy, it suggested that MB50 had a direct effect on the intracellular substrate, although little influence on the membrane, whereas H80 cells showed visible damage to cell membranes. However, it was recognized that PI intensity in MB50 cells was great than that in NT, H50 and H80 cells, and that the amount of nucleic acid and protein leaked from H80 cells was significantly higher than that of NT, MB50 and H50 cells. Furthermore, the enzyme inactivation efficiency in MB50 cells was the same as in H80 cells. These results estimate that inactivation of S. pastorianus by two-stage MB-CO2 was due to actions of MB-CO2 on the cell membrane and the intracellular substrate such as enzyme inactivation.
