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Under carbon source transitions, the intracellular pH of Saccharomyces cerevisiae is subject to change. Dynamics in pH modulate the activity of the glycolytic enzymes, resulting in a change in glycolytic flux and ultimately cell growth. To understand how pH affects the global behavior of glycolysis and ethanol fermentation, we measured the activity of the glycolytic and fermentative enzymes in S. cerevisiae under in vivo‐like conditions at different pH. We demonstrate that glycolytic enzymes exhibit differential pH dependencies, and optima, in the pH range observed during carbon source transitions. The forward reaction of GAPDH shows the highest decrease in activity, 83%, during a simulated feast/famine regime upon glucose removal (cytosolic pH drop from 7.1 to 6.4). We complement our biochemical characterization of the glycolytic enzymes by fitting the Vmax to the progression curves of product formation or decay over time. The fitting analysis shows that the observed changes in enzyme activities require changes in Vmax, but changes in Km cannot be excluded. Our study highlights the relevance of pH as a key player in metabolic regulation and provides a large set of quantitative data that can be explored to improve our understanding of metabolism in dynamic environments. [ABSTRACT FROM AUTHOR]

The yeast Saccharomyces cerevisiae, widely used for ethanol production, is one of the best-understood biological systems. Diploid strains of S. cerevisiae are preferred for industrial use due to the better fermentation efficiency, in terms of vitality and endurance as compared to those of haploid strains. Whole-genome duplications is known to promote adaptive mutations in microorganisms, and allelic variations considerably contribute to the product composition in ethanol fermentation. Although fermentation can be regulated using various strains of yeast, it is quite difficult to make fine adjustment of each component in final products. In this study, we demonstrate the use of polyploids with varying gene dosage (the number of copies of a particular gene present in a genome) in the regulation of ethanol fermentation. Ethyl caproate is one of the major flavouring agents in a Japanese alcoholic beverage called sake. A point mutation in FAS2 encoding the α subunit of fatty acid synthetase induces an increase in the amount of caproic acid, a precursor of ethyl caproate. Using the FAS2 as a model, we generated and evaluated yeast strains with varying mutant gene dosage. We demonstrated the possibility to increase mutant gene dosage via loss of heterozygosity in diploid and tetraploid strains. Productivity of ethyl caproate gradually increased with mutant gene dosage among tetraploid strains. This approach can potentially be applied to a variety of yeast strain development via growth-based screening., (© 2020 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

The capacity of some yeasts to extract energy from single sugars, generating CO2 and ethanol (=fermentation), even in the presence of oxygen, is known as the Crabtree effect. This phenomenon represents an important adaptation as it allowed the utilization of the ecological niche given by modern fruits, an abundant source of food that emerged in the terrestrial environment in the Cretaceous. However, identifying the evolutionary events that triggered fermentative capacity in Crabtree‐positive species is challenging, as microorganisms do not leave fossil evidence. Thus, key innovations should be inferred based only on traits measured under culture conditions. Here, we reanalyzed data from a common garden experiment where several proxies of fermentative capacity were recorded in Crabtree‐positive and Crabtree‐negative species, representing yeast phylogenetic diversity. In particular, we applied the "lasso‐OU" algorithm which detects points of adaptive shifts, using traits that are proxies of fermentative performance. We tested whether multiple events or a single event explains the actual fermentative capacity of yeasts. According to the lasso‐OU procedure, evolutionary changes in the three proxies of fermentative capacity that we considered (i.e., glycerol production, ethanol yield, and respiratory quotient) are consistent with a single evolutionary episode (a whole‐genomic duplication, WGD), instead of a series of small genomic rearrangements. Thus, the WGD appears as the key event behind the diversification of fermentative yeasts, which by increasing gene dosage, and maximized their capacity of energy extraction for exploiting the new ecological niche provided by single sugars. [ABSTRACT FROM AUTHOR]

Lignocellulosic hydrolysates contain acetic acid derived from hemicellulose. Acetate released during acid pretreatment interferes with enzymatic saccharification of the biomass and inhibits yeast fermentation, caused by cellular oxidation and stress. This study used alkaline deacetylation prior to acid pretreatment and evaluated its influence on the fermentation performance of Saccharomyces cerevisiae using two different lignocellulosic biomasses, hemp (Cannabis sativa) and kenaf (Hibiscus cannabinus). They are valuable for biofuel production. The maximal acetate removal with minimal sugar loss was obtained with 1% (w/v) NaOH treatment for both biomasses. Deacetylation pretreatment under this condition was effective both in enzymatic saccharification and simultaneous saccharification and fermentation. This resulted in improved ethanol production by xylose-consuming yeast (S. cerevisiae SR8) by removing 4.7 g/L of acetate from hemp and 6.3 g/L from kenaf, thus resulting in the ethanol productivities of 0.79 and 0.67 g/L-h from hemp and kenaf, respectively. This suggests that strategies to overcome acetate inhibition are necessary to efficiently use the lignocellulosic biomass as a source for bioenergy production. [ABSTRACT FROM AUTHOR]

A goal in insect–microbe ecology is to understand the mechanisms regulating species associations and mutualistic interactions.The spotted wing drosophila Drosophila suzukii develops in ripening fruit, unlike other drosophilids that typically feed on overripe fruit, and is associated with the yeast Hanseniaspora uvarum.We hypothesized that D. suzukii and H. uvarum engage in niche construction leading to a mutualistic relation, facilitating the exploitation of fruit and berries as larval substrate.We show that H. uvarum proliferates on both ripe and on unripe raspberries, mediates attraction of D. suzukii larvae and adult flies, enhances egg‐laying in mated females and is a sufficient food substrate to support larval development. Moreover, H. uvarum suppresses the antagonistic grey mould, Botrytis cinerea in collaboration with D. suzukii larvae, and produces less ethanol than baker's yeast. H. uvarum thus creates favourable conditions for D. suzukii larval development, which is susceptible to ethanol and grey mould.D. suzukii, on the other hand, vectors H. uvarum to suitable substrates such as raspberries, where larval feeding activity enhances growth of H. uvarum. Larval feeding also helps to suppress B. cinerea, which otherwise outcompetes H. uvarum on raspberry, in the absence of fly larvae.In conclusion, H. uvarum enhances D. suzukii larval development on unripe berries, and D. suzukii promotes H. uvarum dispersal and growth on berries. Yeast and fly modify their shared habitat in reciprocal niche construction and mutual interaction. Read the free Plain Language Summary for this article on the Journal blog. [ABSTRACT FROM AUTHOR]

Abstract: Enzymology tends to focus on highly specific effects of substrates, allosteric modifiers, and products occurring at low concentrations, because these are most informative about the enzyme’s catalytic mechanism. We hypothesized that at relatively high in vivo concentrations, important molecular monitors of the state of living cells, such as ATP, affect multiple enzymes of the former and that these interactions have gone unnoticed in enzymology. We test this hypothesis in terms of the effect that ATP, ADP, and AMP might have on the major free-energy delivering pathway of the yeast Saccharomyces cerevisiae. Assaying cell-free extracts, we collected a comprehensive set of quantitative kinetic data concerning the enzymes of the glycolytic and the ethanol fermentation pathways. We determined systematically the extent to which the enzyme activities depend on the concentrations of the adenine nucleotides. We found that the effects of the adenine nucleotides on enzymes catalysing reactions in which they are not directly involved as substrate or product, are substantial. This includes effects on the Michaelis–Menten constants, adding new perspective on these, 100years after their introduction. [Copyright &y& Elsevier]

Lignocellulosic biomass has considerable potential for the production of fuels and chemicals as a promising alternative to conventional fossil fuels. However, the bioconversion of lignocellulosic biomass to desired products must be improved to reach economic viability. One of the main technical hurdles is the presence of inhibitors in biomass hydrolysates, which hampers the bioconversion efficiency by biorefinery microbial platforms such as Saccharomyces cerevisiae in terms of both production yields and rates. In particular, acetic acid, a major inhibitor derived from lignocellulosic biomass, severely restrains the performance of engineered xylose‐utilizing S. cerevisiae strains, resulting in decreased cell growth, xylose utilization rate, and product yield. In this study, the robustness of XUSE, one of the best xylose‐utilizing strains, was improved for the efficient conversion of lignocellulosic biomass into bioethanol under the inhibitory condition of acetic acid stress. Through adaptive laboratory evolution, we successfully developed the evolved strain XUSAE57, which efficiently converted xylose to ethanol with high yields of 0.43–0.50 g ethanol/g xylose even under 2–5 g/L of acetic stress. XUSAE57 not only achieved twofold higher ethanol yields but also improved the xylose utilization rate by more than twofold compared to those of XUSE in the presence of 4 g/L of acetic acid. During fermentation of lignocellulosic hydrolysate, XUSAE57 simultaneously converted glucose and xylose with the highest ethanol yield reported to date (0.49 g ethanol/g sugars). This study demonstrates that the bioconversion of lignocellulosic biomass by an engineered strain could be significantly improved through adaptive laboratory evolution for acetate tolerance, which could help realize the development of an economically feasible lignocellulosic biorefinery to produce fuels and chemicals. [ABSTRACT FROM AUTHOR]

To make the bioethanol conversion process economically viable, improving solids loading (>15% [w/v]) in different unit operations is crucial. In this study, alkali pretreatment of sugarcane bagasse was carried out at high solids loading. Temperature and pH were optimised via batch simultaneous saccharification and fermentation (SSF) experiments using Saccharomyces cerevisiae Y‐2034 or Kluyveromyces marxianus NCYC‐587. The effects of enzyme addition mode and solids loading on ethanol production were studied in fed‐batch SSF experiments. It was demonstrated that the optimised pH and temperature for both S. cerevisiae Y‐2034 and K. marxianus NCYC‐587 were 5.2 and 37°C. The mode of enzyme addition "one‐time dose" improved ethanol productivity better than the "batch addition" mode. With fresh substrates added, solids loading strongly increased from 19% to 33% (w/v), corresponded to a small decline in the ethanol yield. At the final solids loading of 33% (w/v), the highest ethanol concentration of 75.57 ± 2.14 g/L was obtained, with ethanol productivity of 0.63 ± 0.02 g/(L h) and a yield of 66.17 ± 1.87%. Sugarcane bagasse was pretreated with alkali solutions at high solids loading. Fed‐batch simultaneous saccharification and fermentation experiments were conducted at three high solids loadings. The highest ethanol concentration of 75.57 g/L was obtained. [ABSTRACT FROM AUTHOR]

Due to its fast‐growing and high carbohydrate content, Napier grass has a great potential to be chosen as a raw material for renewable energy production. The Napier grass after different pretreatments was tested for simultaneous saccharification and fermentation (SSF) with dried yeast (Saccharomyces cerevisiae) and cellulase (CTec2) to produce ethanol. For alkaline pretreatment, the grass was incubated with 10% NaOH at a ratio of 1:20 (w/v) at 90°C for 1 hour. For dilute acid pretreatment, the grass was soaked with 1% H2SO4 at a ratio of 1:10 (w/v) and incubated at 120°C for 1 hour. For the two‐stage pretreatment, the resultant solid from dilute acid pretreatment was further treated with 2% NaOH (1:10 w/v) at 80°C for 6 hours. After enzymatic saccharification at 50°C and pH 5.0 for 96 hours using CTec2, yields of glucose from three pretreated Napier grass samples (10% water‐insoluble solids, WIS) were 75.4 ± 2.9%, 55.4 ± 6.5%, and 76.4 ± 5.0%. Using 10% WIS of alkaline‐pretreated Napier grass biomass as the substrate, the 72‐hour SSF led to an ethanol yield of 86.6 ± 3.4%. On the basis of the dried biomass of Napier grass as the raw material, the ethanol yield could reach 0.143 ± 0.006 (g/g). Napier grass biomass could be effectively treated by alkaline, dilute acid, or two‐stage pretreatment methods to remove non‐cellulosic components to some extent. Alkaline pretreatment was found to be superior to dilute acid and two‐stage pretreatment methods, based on ethanol yields obtained under similar SSF conditions. Napier grass was pretreated by alkaline, dilute acid, and two‐stage methods. Highest ethanol yield was obtained from alkaline pretreated Napier grass. An ethanol yield of 0.143 g/g‐dried raw material of Napier grass was achieved by simultaneous saccharification and fermentation (SSF). [ABSTRACT FROM AUTHOR]

Dynamic flux balance analysis was utilized to simulate the metabolic behaviour of initially fully respirative and respirofermentative steady-state cultures of Saccharomyces cerevisiae during sudden oxygen depletion. The hybrid model for the dynamic flux balance analysis included a stoichiometric genome-scale metabolic model as a static part and dynamic equations for the uptake of glucose and the cessation of respirative metabolism. The yeast consensus genome-scale metabolic model [Herrgård MJ et al. (2008) Nat Biotechnol 26, 1155-1160; Dobson PD et al. (2010) BMC Syst Biol 4, 145] was refined with respect to oxygen-dependent energy metabolism and further modified to reflect S. cerevisiae anabolism in the absence of oxygen. Dynamic flux balance analysis captured well the essential features of the dynamic metabolic behaviour of S. cerevisiae during adaptation to anaerobiosis. Modelling and simulation enabled the identification of short time-scale flux distribution dynamics under the transition to anaerobic metabolism, during which the specific growth rate was reduced, as well as longer time-scale process dynamics when the specific growth rate recovered. Expression of the metabolic genes was set into the context of the identified dynamics. Metabolic gene expression responses associated with the specific growth rate and with the cessation of respirative metabolism were distinguished. [ABSTRACT FROM AUTHOR]

The growth of Saccharomyces cerevisiae cells under aerobic conditions, in the presence of an energy-rich source, leads to production of an excess of NAD(P)H. Since the redox balance must be maintained, it has been postulated that NAD(P)H reoxidation is accelerated by the activation of energy-dissipating reactions, which would, in turn, explain the low growth efficiencies observed. It has been demonstrated already in S. cerevisiae cultures that these putative energy-dissipating reactions are stimulated both by oxygen and high cytosolic ATP levels. In this paper, we show that ATP induces a proton-permeability pathway in mitochondria at concentrations which are within the physiological range, as revealed both from the ATP stimulation of respiration and from the induction of H+-dependent swelling. We also demonstrate that phosphate acts as a competitive inhibitor of the nucleotide, and since activation is observed even in the presence of atractylate, we postulate that the ATP-binding site is located in the outer face of the mitochondrial inner membrane. [ABSTRACT FROM AUTHOR]

After whole-genome duplication (WGD), deletions return most loci to single copy. However, duplicate loci may survive through selection for increased dosage. Here, we show how the WGD increased copy number of some glycolytic genes could have conferred an almost immediate selective advantage to an ancestor of Saccharomyces cerevisiae, providing a rationale for the success of the WGD. We propose that the loss of other redundant genes throughout the genome resulted in incremental dosage increases for the surviving duplicated glycolytic genes. This increase gave post-WGD yeasts a growth advantage through rapid glucose fermentation; one of this lineage's many adaptations to glucose-rich environments. Our hypothesis is supported by data from enzyme kinetics and comparative genomics. Because changes in gene dosage follow directly from post-WGD deletions, dosage selection can confer an almost instantaneous benefit after WGD, unlike neofunctionalization or subfunctionalization, which require specific mutations. We also show theoretically that increased fermentative capacity is of greatest advantage when glucose resources are both large and dense, an observation potentially related to the appearance of angiosperms around the time of WGD. [ABSTRACT FROM AUTHOR]