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Jack T Pronk - One of the best experts on this subject based on the ideXlab platform.
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under pressure Evolutionary Engineering of yeast strains for improved performance in fuels and chemicals production
Current Opinion in Biotechnology, 2018Co-Authors: Robert Mans, Jean-marc Daran, Jack T PronkAbstract:Evolutionary Engineering, which uses laboratory evolution to select for industrially relevant traits, is a popular strategy in the development of high-performing yeast strains for industrial production of fuels and chemicals. By integrating whole-genome sequencing, bioinformatics, classical genetics and genome-editing techniques, Evolutionary Engineering has also become a powerful approach for identification and reverse Engineering of molecular mechanisms that underlie industrially relevant traits. New techniques enable acceleration of in vivo mutation rates, both across yeast genomes and at specific loci. Recent studies indicate that phenotypic trade-offs, which are often observed after evolution under constant conditions, can be mitigated by using dynamic cultivation regimes. Advances in research on synthetic regulatory circuits offer exciting possibilities to extend the applicability of Evolutionary Engineering to products of yeasts whose synthesis requires a net input of cellular energy.
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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, Jack T Pronk, Marcel Van Den Broek, Brian Gibson, 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, Jack T Pronk, Marcel Van Den Broek, Brian Gibson, 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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Evolutionary Engineering to enhance starter culture performance in food fermentations
Current Opinion in Biotechnology, 2015Co-Authors: Herwig Bachmann, Jack T Pronk, Michiel Kleerebezem, Bas TeusinkAbstract:Microbial starter cultures are essential for consistent product quality and functional properties such as flavor, texture, pH or the alcohol content of various fermented foods. Strain improvement programs to achieve desired properties in starter cultures are diverse, but developments in next-generation sequencing lead to an increased interest in Evolutionary Engineering of desired phenotypes. We here discuss recent developments of strain selection protocols and how computational approaches can assist such experimental design. Furthermore the analysis of evolved phenotypes and possibilities with complex consortia are highlighted. Studies carried out with mainly yeast and lactic acid bacteria demonstrate the power of Evolutionary Engineering to deliver strains with novel phenotypes as well as insight into underlying mechanisms.
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Evolutionary Engineering of a glycerol 3 phosphate dehydrogenase negative acetate reducing saccharomyces cerevisiae strain enables anaerobic growth at high glucose concentrations
Microbial Biotechnology, 2014Co-Authors: Victor Guadalupemedina, Jack T Pronk, Robert Mans, Bart Oud, Benjamin Metz, Charlotte M Van Der Graaf, Antonius J A Van MarisAbstract:Glycerol production by Saccharomyces cerevisiae, which is required for redox-cofactor balancing in anaerobic cultures, causes yield reduction in industrial bioethanol production. Recently, glycerol formation in anaerobic S.?cerevisiae cultures was eliminated by expressing Escherichia coli (acetylating) acetaldehyde dehydrogenase (encoded by mhpF) and simultaneously deleting the GPD1 and GPD2 genes encoding glycerol-3-phosphate dehydrogenase, thus coupling NADH reoxidation to reduction of acetate to ethanol. Gpd– strains are, however, sensitive to high sugar concentrations, which complicates industrial implementation of this metabolic Engineering concept. In this study, laboratory evolution was used to improve osmotolerance of a Gpd– mhpF-expressing S.?cerevisiae strain. Serial batch cultivation at increasing osmotic pressure enabled isolation of an evolved strain that grew anaerobically at 1?M glucose, at a specific growth rate of 0.12?h?1. The evolved strain produced glycerol at low concentrations (0.64?±?0.33?g?l?1). However, these glycerol concentrations were below 10% of those observed with a Gpd+ reference strain. Consequently, the ethanol yield on sugar increased from 79% of the theoretical maximum in the reference strain to 92% for the evolved strains. Genetic analysis indicated that osmotolerance under aerobic conditions required a single dominant chromosomal mutation, and one further mutation in the plasmid-borne mhpF gene for anaerobic growth.
Sun-mi Lee - One of the best experts on this subject based on the ideXlab platform.
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xylan catabolism is improved by blending bioprospecting and metabolic pathway Engineering in saccharomyces cerevisiae
Biotechnology Journal, 2015Co-Authors: Sun-mi Lee, Taylor Jellison, Hal S. AlperAbstract:Complete utilization of all available carbon sources in lignocellulosic biomass still remains a challenge in Engineering Saccharomyces cerevisiae. Even with efficient heterologous xylose catabolic pathways, S. cerevisiae is unable to utilize xylose in lignocellulosic biomass unless xylan is depolymerized to xylose. Here we demonstrate that a blended bioprospecting approach along with pathway Engineering and Evolutionary Engineering can be used to improve xylan catabolism in S. cerevisiae. Specifically, we perform whole genome sequencing-based bioprospecting of a strain with remarkable pentose catabolic potential that we isolated and named Ustilago bevomyces. The heterologous expression of xylan catabolic genes enabled S. cerevisiae to grow on xylan as a single carbon source in minimal medium. A combination of bioprospecting and metabolic pathway evolution demonstrated that the xylan catabolic pathway could be further improved. Ultimately, Engineering efforts were able to achieve xylan conversion into ethanol of up to 0.22 g/L on minimal medium compositions with xylan. This pathway provides a novel starting point for improving lignocellulosic conversion by yeast.
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systematic and Evolutionary Engineering of a xylose isomerase based pathway in saccharomyces cerevisiae for efficient conversion yields
Biotechnology for Biofuels, 2014Co-Authors: Sun-mi Lee, Taylor Jellison, Hal S. AlperAbstract:Background Efficient xylose fermentation by yeast would improve the economical and sustainable nature of biofuels production from lignocellulosic biomass. However, the efficiency of xylose fermentation by the yeast Saccharomyces cerevisiae is suboptimal, especially in conversion yield, despite decades of research. Here, we present an improved performance of S. cerevisiae in xylose fermentation through systematic and Evolutionary Engineering approaches.
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systematic and Evolutionary Engineering of a xylose isomerase based pathway in saccharomyces cerevisiae for efficient conversion yields
Biotechnology for Biofuels, 2014Co-Authors: Sun-mi Lee, Taylor Jellison, Hal S. AlperAbstract:Efficient xylose fermentation by yeast would improve the economical and sustainable nature of biofuels production from lignocellulosic biomass. However, the efficiency of xylose fermentation by the yeast Saccharomyces cerevisiae is suboptimal, especially in conversion yield, despite decades of research. Here, we present an improved performance of S. cerevisiae in xylose fermentation through systematic and Evolutionary Engineering approaches. The Engineering of S. cerevisiae harboring xylose isomerase-based pathway significantly improved the xylose fermentation performance without the need for intensive downstream pathway Engineering. This strain contained two integrated copies of a mutant xylose isomerase, gre3 and pho13 deletion and XKS1 and S. stipitis tal1 overexpression. This strain was subjected to rapid adaptive evolution to yield the final, evolved strain (SXA-R2P-E) which could efficiently convert xylose to ethanol with a yield of 0.45 g ethanol/g xylose, the highest yield reported to date. The xylose consumption and ethanol production rates, 0.98 g xylose g cell−1 h−1 and 0.44 g ethanol g cell−1 h−1, respectively, were also among the highest reported. During this process, the positive effect of a pho13 deletion was identified for a xylose isomerase-containing strain and resulted in up to an 8.2-fold increase in aerobic growth rate on xylose. Moreover, these results demonstrated that low inoculum size and the cell transfer at exponential phase was found to be the most effective adaptation strategy during a batch culture adaptation process. These results suggest that the xylose isomerase pathway should be the pathway of choice for efficient xylose fermentation in S. cerevisiae as it can outperform strains with the oxidoreductase pathway in terms of yield and ethanol production and xylose consumption rates. Consequently, the strain developed in this study could significantly improve the prospect of biofuels production from lignocellulosic biomass.
Zhen Cai - One of the best experts on this subject based on the ideXlab platform.
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genome replication Engineering assisted continuous evolution greace to improve microbial tolerance for biofuels production
Biotechnology for Biofuels, 2013Co-Authors: Guodong Luan, Zhen CaiAbstract:Background Microbial production of biofuels requires robust cell growth and metabolism under tough conditions. Conventionally, such tolerance phenotypes were engineered through Evolutionary Engineering using the principle of “Mutagenesis followed-by Selection”. The iterative rounds of mutagenesis-selection and frequent manual interventions resulted in discontinuous and inefficient strain improvement processes. This work aimed to develop a more continuous and efficient Evolutionary Engineering method termed as “Genome Replication Engineering Assisted Continuous Evolution” (GREACE) using “Mutagenesis coupled-with Selection” as its core principle.
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genome replication Engineering assisted continuous evolution greace to improve microbial tolerance for biofuels production
Biotechnology for Biofuels, 2013Co-Authors: Guodong Luan, Zhen CaiAbstract:Microbial production of biofuels requires robust cell growth and metabolism under tough conditions. Conventionally, such tolerance phenotypes were engineered through Evolutionary Engineering using the principle of “Mutagenesis followed-by Selection”. The iterative rounds of mutagenesis-selection and frequent manual interventions resulted in discontinuous and inefficient strain improvement processes. This work aimed to develop a more continuous and efficient Evolutionary Engineering method termed as “Genome Replication Engineering Assisted Continuous Evolution” (GREACE) using “Mutagenesis coupled-with Selection” as its core principle. The core design of GREACE is to introduce an in vivo continuous mutagenesis mechanism into microbial cells by introducing a group of genetically modified proofreading elements of the DNA polymerase complex to accelerate the evolution process under stressful conditions. The genotype stability and phenotype heritability can be stably maintained once the genetically modified proofreading element is removed, thus scarless mutants with desired phenotypes can be obtained. Kanamycin resistance of E. coli was rapidly improved to confirm the concept and feasibility of GREACE. Intrinsic mechanism analysis revealed that during the continuous evolution process, the accumulation of genetically modified proofreading elements with mutator activities endowed the host cells with enhanced adaptation advantages. We further showed that GREACE can also be applied to engineer n-butanol and acetate tolerances. In less than a month, an E. coli strain capable of growing under an n-butanol concentration of 1.25% was isolated. As for acetate tolerance, cell growth of the evolved E. coli strain increased by 8-fold under 0.1% of acetate. In addition, we discovered that adaptation to specific stresses prefers accumulation of genetically modified elements with specific mutator strengths. We developed a novel GREACE method using “Mutagenesis coupled-with Selection” as core principle. Successful isolation of E. coli strains with improved n-butanol and acetate tolerances demonstrated the potential of GREACE as a promising method for strain improvement in biofuels production.
Antonius J A Van Maris - One of the best experts on this subject based on the ideXlab platform.
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Evolutionary Engineering of a glycerol 3 phosphate dehydrogenase negative acetate reducing saccharomyces cerevisiae strain enables anaerobic growth at high glucose concentrations
Microbial Biotechnology, 2014Co-Authors: Victor Guadalupemedina, Jack T Pronk, Robert Mans, Bart Oud, Benjamin Metz, Charlotte M Van Der Graaf, Antonius J A Van MarisAbstract:Glycerol production by Saccharomyces cerevisiae, which is required for redox-cofactor balancing in anaerobic cultures, causes yield reduction in industrial bioethanol production. Recently, glycerol formation in anaerobic S.?cerevisiae cultures was eliminated by expressing Escherichia coli (acetylating) acetaldehyde dehydrogenase (encoded by mhpF) and simultaneously deleting the GPD1 and GPD2 genes encoding glycerol-3-phosphate dehydrogenase, thus coupling NADH reoxidation to reduction of acetate to ethanol. Gpd– strains are, however, sensitive to high sugar concentrations, which complicates industrial implementation of this metabolic Engineering concept. In this study, laboratory evolution was used to improve osmotolerance of a Gpd– mhpF-expressing S.?cerevisiae strain. Serial batch cultivation at increasing osmotic pressure enabled isolation of an evolved strain that grew anaerobically at 1?M glucose, at a specific growth rate of 0.12?h?1. The evolved strain produced glycerol at low concentrations (0.64?±?0.33?g?l?1). However, these glycerol concentrations were below 10% of those observed with a Gpd+ reference strain. Consequently, the ethanol yield on sugar increased from 79% of the theoretical maximum in the reference strain to 92% for the evolved strains. Genetic analysis indicated that osmotolerance under aerobic conditions required a single dominant chromosomal mutation, and one further mutation in the plasmid-borne mhpF gene for anaerobic growth.
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batch and continuous culture based selection strategies for acetic acid tolerance in xylose fermenting saccharomyces cerevisiae
Fems Yeast Research, 2011Co-Authors: Jeremiah Wright, Eleonora Bellissimi, Jack T Pronk, Erik De Hulster, Andreas Wagner, Antonius J A Van MarisAbstract:Acetic acid tolerance of Saccharomyces cerevisiae is crucial for the production of bioethanol and other bulk chemicals from lignocellulosic plant-biomass hydrolysates, especially at a low pH. This study explores two Evolutionary Engineering strategies for the improvement of acetic acid tolerance of the xylose-fermenting S. cerevisiae RWB218, whose anaerobic growth on xylose at pH 4 is inhibited at acetic acid concentrations >1 g L−1: (1) sequential anaerobic, batch cultivation (pH 4) at increasing acetic acid concentrations and (2) prolonged anaerobic continuous cultivation without pH control, in which acidification by ammonium assimilation generates selective pressure for acetic acid tolerance. After c. 400 generations, the sequential-batch and continuous selection cultures grew on xylose at pH≤4 with 6 and 5 g L−1 acetic acid, respectively. In the continuous cultures, the specific xylose-consumption rate had increased by 75% to 1.7 g xylose g−1 biomass h−1. After storage of samples from both selection experiments at −80 °C and cultivation without acetic acid, they failed to grow on xylose at pH 4 in the presence of 5 g L−1 acetic acid. Characterization in chemostat cultures with linear acetic acid gradients demonstrated an acetate-inducible acetic acid tolerance in samples from the continuous selection protocol.
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metabolome transcriptome and metabolic flux analysis of arabinose fermentation by engineered saccharomyces cerevisiae
Metabolic Engineering, 2010Co-Authors: Wouter H Wisselink, Jack T Pronk, Chiara Cipollina, Bart Oud, Barbara Crimi, Joseph J Heijnen, Antonius J A Van MarisAbstract:One of the challenges in strain improvement by Evolutionary Engineering is to subsequently determine the molecular basis of the improved properties that were enriched from the natural genetic variation during the selective conditions. This study focuses on Saccharomyces cerevisiae IMS0002 which, after metabolic and Evolutionary Engineering, ferments the pentose sugar arabinose. Glucose- and arabinose-limited anaerobic chemostat cultures of IMS0002 and its non-evolved ancestor were subjected to transcriptome analysis, intracellular metabolite measurements and metabolic flux analysis. Increased expression of the GAL-regulon and deletion of GAL2 in IMS0002 confirmed that the galactose transporter is essential for growth on arabinose. Elevated intracellular concentrations of pentose-phosphate-pathway intermediates and upregulation of TKL2 and YGR043c (encoding transketolase and transaldolase isoenzymes) suggested an involvement of these genes in flux-controlling reactions in arabinose fermentation. Indeed, deletion of these genes in IMS0002 caused a 21% reduction of the maximum specific growth rate on arabinose.
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novel Evolutionary Engineering approach for accelerated utilization of glucose xylose and arabinose mixtures by engineered saccharomyces cerevisiae strains
Applied and Environmental Microbiology, 2009Co-Authors: Wouter H Wisselink, Maurice J Toirkens, Jack T Pronk, Antonius J A Van MarisAbstract:Lignocellulosic feedstocks are thought to have great economic and environmental significance for future biotechnological production processes. For cost-effective and efficient industrial processes, complete and fast conversion of all sugars derived from these feedstocks is required. Hence, simultaneous or fast sequential fermentation of sugars would greatly contribute to the efficiency of production processes. One of the main challenges emerging from the use of lignocellulosics for the production of ethanol by the yeast Saccharomyces cerevisiae is efficient fermentation of D-xylose and L-arabinose, as these sugars cannot be used by natural S. cerevisiae strains. In this study, we describe the first engineered S. cerevisiae strain (strain IMS0003) capable of fermenting mixtures of glucose, xylose, and arabinose with a high ethanol yield (0.43 g g–1 of total sugar) without formation of the side products xylitol and arabinitol. The kinetics of anaerobic fermentation of glucose-xylose-arabinose mixtures were greatly improved by using a novel Evolutionary Engineering strategy. This strategy included a regimen consisting of repeated batch cultivation with repeated cycles of consecutive growth in three media with different compositions (glucose, xylose, and arabinose; xylose and arabinose; and only arabinose) and allowed rapid selection of an evolved strain (IMS0010) exhibiting improved specific rates of consumption of xylose and arabinose. This evolution strategy resulted in a 40% reduction in the time required to completely ferment a mixture containing 30 g liter–1 glucose, 15 g liter–1 xylose, and 15 g liter–1 arabinose.
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Engineering of saccharomyces cerevisiae for efficient anaerobic alcoholic fermentation of l arabinose
Applied and Environmental Microbiology, 2007Co-Authors: Wouter H Wisselink, Johannes P. Van Dijken, Maurice J Toirkens, M Del Rosario Franco Berriel, Aaron Adriaan Winkler, Jack T Pronk, Antonius J A Van MarisAbstract:For cost-effective and efficient ethanol production from lignocellulosic fractions of plant biomass, the conversion of not only major constituents, such as glucose and xylose, but also less predominant sugars, such as L-arabinose, is required. Wild-type strains of Saccharomyces cerevisiae, the organism used in industrial ethanol production, cannot ferment xylose and arabinose. Although metabolic and Evolutionary Engineering has enabled the efficient alcoholic fermentation of xylose under anaerobic conditions, the conversion of L-arabinose into ethanol by engineered S. cerevisiae strains has previously been demonstrated only under oxygen-limited conditions. This study reports the first case of fast and efficient anaerobic alcoholic fermentation of L-arabinose by an engineered S. cerevisiae strain. This fermentation was achieved by combining the expression of the structural genes for the L-arabinose utilization pathway of Lactobacillus plantarum, the overexpression of the S. cerevisiae genes encoding the enzymes of the nonoxidative pentose phosphate pathway, and extensive Evolutionary Engineering. The resulting S. cerevisiae strain exhibited high rates of arabinose consumption (0.70 g h–1 g [dry weight]–1) and ethanol production (0.29 g h–1 g [dry weight]–1) and a high ethanol yield (0.43 g g–1) during anaerobic growth on L-arabinose as the sole carbon source. In addition, efficient ethanol production from sugar mixtures containing glucose and arabinose, which is crucial for application in industrial ethanol production, was achieved.
Jean-marc Daran - One of the best experts on this subject based on the ideXlab platform.
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under pressure Evolutionary Engineering of yeast strains for improved performance in fuels and chemicals production
Current Opinion in Biotechnology, 2018Co-Authors: Robert Mans, Jean-marc Daran, Jack T PronkAbstract:Evolutionary Engineering, which uses laboratory evolution to select for industrially relevant traits, is a popular strategy in the development of high-performing yeast strains for industrial production of fuels and chemicals. By integrating whole-genome sequencing, bioinformatics, classical genetics and genome-editing techniques, Evolutionary Engineering has also become a powerful approach for identification and reverse Engineering of molecular mechanisms that underlie industrially relevant traits. New techniques enable acceleration of in vivo mutation rates, both across yeast genomes and at specific loci. Recent studies indicate that phenotypic trade-offs, which are often observed after evolution under constant conditions, can be mitigated by using dynamic cultivation regimes. Advances in research on synthetic regulatory circuits offer exciting possibilities to extend the applicability of Evolutionary Engineering to products of yeasts whose synthesis requires a net input of cellular energy.
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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, Jack T Pronk, Marcel Van Den Broek, Brian Gibson, 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, Jack T Pronk, Marcel Van Den Broek, Brian Gibson, 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
