Variation in the modality of a yeast signaling pathway is mediated by a single regulator

Bimodal gene expression by genetically identical cells is a pervasive feature of signaling networks, and has been suggested to allow organisms to hedge their "bets" in uncertain conditions. In the galactose-utilization (GAL) pathway of Saccharomyces cerevisiae, gene induction is unimodal or bimodal depending on natural genetic variation and pre-induction conditions. Here, we find that this variation in modality arises from regulation of two features of the pathway response: the fraction of cells that show induction, and their level of expression. GAL3, the galactose sensor, controls the fraction of induced cells, and titrating its expression is sufficient to control modality; moreover, all the observed differences in modality between different pre-induction conditions and amongst natural isolates can be explained by changes in GAL3's regulation and activity. The ability to switch modality by tuning the activity of a single protein may allow rapid adaptation of bet hedging to maximize fitness in complex environments.

Effect of deregulation of repressor-specific carbon catabolite repression on carbon source consumption in Escherichia coli

Escherichia coli has been used as a host to construct the cell factory for biobased production of chemicals from renewable feedstocks. Because galactose is found in marine biomass as a major component, the strategy for galactose utilization in E. coli has been gained more attention. Although galactose and glucose co-fermentation has been reported using the engineered E. coli strain, few reports have covered fermentation supplemented with galactose as a sole carbon source in the mutant lacking the repressor-specific carbon catabolite repression (CCR). Here, we report the effects of the deregulation of the repressor-specific CCR (galR− and galS−) in fermentation supplemented with galactose as a sole carbon source, using the engineered E. coli strains. In the fermentation using the galR− and galS− double mutant (GR2 strain), an increase of rates in sugar consumption and cell growth was observed compared to the parent strain. In the glucose fermentation, wild-type W3110 and its mutant GR2 and GR2PZ (galR−, galS−, pfkA−, and zwf−) consumed sugar at a higher rate than those values obtained from galactose fermentation. However, the GR2P strain (galR−, galS−, and pfkA−) showed no difference between fermentations using glucose and galactose as a sole carbon source. This study provides essential information for galactose fermentation using the CCR-deregulated E. coli strains.

Transcriptional Profiling of Myceliophthora thermophila on Galactose and Metabolic Engineering for Improved Galactose Utilization

Efficient biological conversion of all sugars from lignocellulosic biomass is necessary for the cost-effective production of biofuels and commodity chemicals. Galactose is one of the most abundant sugar in many hemicelluloses, and it will be important to capture this carbon for an efficient bioconversion process of plant biomass. Thermophilic fungus Myceliophthora thermophila has been used as a cell factory to produce biochemicals directly from renewable polysaccharides. In this study, we draw out the two native galactose utilization pathways, including the Leloir pathway and oxido-reductive pathway, and identify the significance and contribution of them, through transcriptional profiling analysis of M. thermophila and its mutants on galactose. We find that galactokinase was necessary for galactose transporter expression, and disruption of galK resulted in decreased galactose utilization. Through metabolic engineering, both galactokinase deletion and galactose transporter overexpression can activate internal the oxido-reductive pathway and improve the consumption rate of galactose. Finally, the heterologous galactose-degradation pathway, De Ley–Doudoroff (DLD) pathway, was successfully integrated into M. thermophila, and the consumption rate of galactose in the engineered strain was increased by 57%. Our study focuses on metabolic engineering for accelerating galactose utilization in a thermophilic fungus that will be beneficial for the rational design of fungal strains to produce biofuels and biochemicals from a variety of feedstocks with abundant galactose.

Ancient balancing selection maintains incompatible versions of the galactose pathway in yeast

Metabolic pathways differ across species but are expected to be similar within a species. We discovered two functional, incompatible versions of the galactose pathway in Saccharomyces cerevisiae. We identified a three-locus genetic interaction for growth in galactose, and used precisely engineered alleles to show that it arises from variation in the galactose utilization genes GAL2, GAL1/10/7, and phosphoglucomutase (PGM1), and that the reference allele of PGM1 is incompatible with the alternative alleles of the other genes. Multiloci balancing selection has maintained the two incompatible versions of the pathway for millions of years. Strains with alternative alleles are found primarily in galactose-rich dairy environments, and they grow faster in galactose but slower in glucose, revealing a trade-off on which balancing selection may have acted.

Gut-derived Flavonifractor species variants are differentially enriched during in vitro incubation with quercetin

Flavonoids are a common component of the human diet with widely reported health-promoting properties. The gut microbiota transforms these compounds affecting the overall metabolic outcome of flavonoid consumption. Flavonoid-degrading bacteria are often studied in pure and mixed cultures but the multiple interactions between quercetin-degraders and the rest of the community have been overlooked. In this study, a comparative metataxonomic analysis of fecal communities supplemented with the flavonoid quercetin led us to identify a potential competitive exclusion interaction between two sequence variants related to the flavonoid-degrading species, Flavonifractor plautii, that belong to the same genus but different species. During incubation of fecal slurries with quercetin, the relative abundance of these two variants was inversely correlated; one variant, ASV_65f4, increased in relative abundance in half of the libraries and the other variant, ASV_a45d, in the other half. This pattern was also observed with 6 additional fecal samples that were transplanted into germ-free mice fed two different diets. Mouse’s diet did not change the pattern of dominance of either variant, and initial relative abundances did not predict which one ended up dominating. Potential distinct metabolic capabilities of these two Flavonifractor-related species were evidenced, as only one variant, ASV_65f4, became consistently enriched in complex communities supplemented with acetate but without quercetin. Genomic comparison analysis of the close relatives of each variant revealed that ASV_65f4 may be an efficient utilizer of ethanolamine which is formed from the phospholipid phosphatidylethanolamine that is abundant in the gut and feces. Other discordant features between ASV_65f4- and ASV_a45d-related groups may be the presence of flagellar and galactose-utilization genes, respectively. Overall, we showed that the Flavonifractor genus harbors variants that present a pattern of negative co-occurrence and that may have different metabolic and morphological traits, whether these differences affect the dynamic of quercetin degradation warrants further investigation.

Repeated horizontal gene transfer of GALactose metabolism genes violates Dollo’s law of irreversible loss

Dollo’s law posits that evolutionary losses are irreversible, thereby narrowing the potential paths of evolutionary change. While phenotypic reversals to ancestral states have been observed, little is known about their underlying genetic causes. The genomes of budding yeasts have been shaped by extensive reductive evolution, such as reduced genome sizes and the losses of metabolic capabilities. However, the extent and mechanisms of trait reacquisition after gene loss in yeasts have not been thoroughly studied. Here, through phylogenomic analyses, we reconstructed the evolutionary history of the yeast galactose utilization pathway and observed widespread and repeated losses of the ability to utilize galactose, which occurred concurrently with the losses of GALactose (GAL) utilization genes. Unexpectedly, we detected multiple galactose-utilizing lineages that were deeply embedded within clades that underwent ancient losses of galactose utilization. We show that at least two, and possibly three, lineages reacquired the GAL pathway via yeast-to-yeast horizontal gene transfer. Our results show how trait reacquisition can occur tens of millions of years after an initial loss via horizontal gene transfer from distant relatives. These findings demonstrate that the losses of complex traits and even whole pathways are not always evolutionary dead-ends, highlighting how reversals to ancestral states can occur.

Repeated horizontal gene transfer of GALactose metabolism genes violates Dollo’s law of irreversible loss

Dollo’s law posits that evolutionary losses are irreversible, thereby narrowing the potential paths of evolutionary change. While phenotypic reversals to ancestral states have been observed, little is known about their underlying genetic causes. The genomes of budding yeasts have been shaped by extensive reductive evolution, such as reduced genome sizes and the losses of metabolic capabilities. However, the extent and mechanisms of trait reacquisition after gene loss in yeasts have not been thoroughly studied. Here, through phylogenomic analyses, we reconstructed the evolutionary history of the yeast galactose utilization pathway and observed widespread and repeated losses of the ability to utilize galactose, which occurred concurrently with the losses of GALactose (GAL) utilization genes. Unexpectedly, we detected three galactose-utilizing lineages that were deeply embedded within clades that underwent ancient losses of galactose utilization. We show that at least two, and possibly three, lineages reacquired the GAL pathway via yeast-to-yeast horizontal gene transfer. Our results show how trait reacquisition can occur tens of millions of years after an initial loss via horizontal gene transfer from distant relatives. These findings demonstrate that the losses of complex traits and even whole pathways are not always evolutionary dead-ends, highlighting how reversals to ancestral states can occur.

Computational study on ratio-sensing in yeast galactose utilization pathway

Metabolic networks undergo gene expression regulation in response to external nutrient signals. In microbes, the synthesis of enzymes that are used to transport and catabolize less preferred carbon sources is repressed in the presence of a preferred carbon source. For most microbes, glucose is a preferred carbon source, and it has long been believed that as long as glucose is present in the environment, the expression of genes related to the metabolism of alternative carbon sources is shut down, due to catabolite repression. However, recent studies have shown that the induction of the galactose (GAL) metabolic network does not solely depend on the exhaustion of glucose. Instead, the GAL genes respond to the external concentration ratio of galactose to glucose, a phenomenon of unknown mechanism that we termed ratio-sensing. Using mathematical modeling, we found that ratio-sensing is a general phenomenon that can arise from competition between two carbon sources for shared transporters, between transcription factors for binding to communal regulatory sequences of the target genes, or a combination of the aforementioned two levels of competition. We analyzed how the parameters describing the competitive interaction influenced ratio-sensing behaviors in each scenario and found that the concatenation of both layers of signal integration can expand the dynamical range of ratio-sensing. Finally, we investigated the influence of circuit topology on ratio-sensing and found that incorporating negative auto-regulation and/or coherent feedforward loop motifs to the basic signal integration unit can tune the sensitivity of the response to the external nutrient signals. Our study not only deepened our understanding of how ratio-sensing is achieved in yeast GAL metabolic regulation, but also elucidated design principles for ratio-sensing signal processing that can be used in other biological settings, such as being introduced into circuit designs for synthetic biology applications.Author summaryMicrobes make sophisticated choices about the uptake and metabolism of nutrients depending on the variety of nutrient choices available to them in their environment. In the well-studied yeast galactose utilization network, a recent study has shown that galactose metabolic genes respond to the external concentration ratio of galactose to glucose. Using computational models, we showed that this type of phenomenon could arise from a competition between galactose and glucose for transporters, a competition between transcription factors for promoters, or a combination of these two mechanisms. We further revealed the controlling parameters that determined the system sensitivity towards competing input signals and that determined the concentration ratio required to induce the metabolic network in each scenario. Combining competition inhibition at both the transporter level and the transcriptional level can enlarge the ratio-sensing regime, resulting a robust signal integration module. We suspect that modules of this kind may be common in many areas of biology.