Ploidy evolution in a wild yeast is linked to an interaction between cell type and metabolism.

Ploidy is an evolutionarily labile trait, and its variation across the tree of life has profound impacts on evolutionary trajectories and life histories. The immediate consequences and molecular causes of ploidy variation on organismal fitness are frequently less clear, although extreme mating type skews in some fungi hint at links between cell type and adaptive traits. Here, we report an unusual recurrent ploidy reduction in replicate populations of the budding yeast Saccharomyces eubayanus experimentally evolved for improvement of a key metabolic trait, the ability to use maltose as a carbon source. We find that haploids have a substantial, but conditional, fitness advantage in the absence of other genetic variation. Using engineered genotypes that decouple the effects of ploidy and cell type, we show that increased fitness is primarily due to the distinct transcriptional program deployed by haploid-like cell types, with a significant but smaller contribution from absolute ploidy. The link between cell-type specification and the carbon metabolism adaptation can be traced to the noncanonical regulation of a maltose transporter by a haploid-specific gene. This study provides novel mechanistic insight into the molecular basis of an environment-cell type fitness interaction and illustrates how selection on traits unexpectedly linked to ploidy states or cell types can drive karyotypic evolution in fungi.

The Brazilian Amazonian rainforest harbors a high diversity of yeasts associated with rotting wood, including many candidates for new yeast species.

This study investigated the diversity of yeast species associated with rotting wood in Brazilian Amazonian rainforests. A total of 569 yeast strains were isolated from rotting wood samples collected in three Amazonian areas (Universidade Federal do Amazonas-Universidade Federal do Amazonas [UFAM], Piquiá, and Carú) in the municipality of Itacoatiara, Amazon state. The samples were cultured in yeast nitrogen base (YNB)-d-xylose, YNB-xylan, and sugarcane bagasse and corncob hemicellulosic hydrolysates (undiluted and diluted 1:2 and 1:5). Sugiyamaella was the most prevalent genus identified in this work, followed by Kazachstania. The most frequently isolated yeast species were Schwanniomyces polymorphus, Scheffersomyces amazonensis, and Wickerhamomyces sp., respectively. The alpha diversity analyses showed that the dryland forest of UFAM was the most diverse area, while the floodplain forest of Carú was the least. Additionally, the difference in diversity between UFAM and Carú was the highest among the comparisons. Thirty candidates for new yeast species were obtained, representing 36% of the species identified and totaling 101 isolates. Among them were species belonging to the clades Spathaspora, Scheffersomyces, and Sugiyamaella, which are recognized as genera with natural xylose-fermenting yeasts that are often studied for biotechnological and ecological purposes. The results of this work showed that rotting wood collected from the Amazonian rainforest is a tremendous source of diverse yeasts, including candidates for new species.

Saccharomycopsis praedatoria sp. nov., a predacious yeast isolated from soil and rotten wood in an Amazonian rainforest biome.

Three yeast isolates were obtained from soil and rotting wood samples collected in an Amazonian rainforest biome in Brazil. Comparison of the intergenic spacer 5.8S region and the D1/D2 domains of the large subunit rRNA gene showed that the isolates represent a novel species of the genus Saccharomycopsis. A tree inferred from the D1/D2 sequences placed the novel species near a subclade containing Saccharomycopsis lassenensis, Saccharomycopsis fermentans, Saccharomycopsis javanensis, Saccharomycopsis babjevae, Saccharomycopsis schoenii and Saccharomycopsis oosterbeekiorum, but with low bootstrap support. In terms of sequence divergence, the novel species had the highest identity in the D1/D2 domains with Saccharomycopsis capsularis, from which it differed by 36 substitutions. In contrast, a phylogenomic analysis based on 1061 single-copy orthologs for a smaller set of Saccharomycopsis species whose whole genome sequences are available indicated that the novel species represented by strain UFMG-CM-Y6991 is phylogenetically closer to Saccharomycopsis fodiens and Saccharomycopsis sp. TF2021a (=Saccharomycopsis phalluae). The novel yeast is homothallic and produces asci with one spheroidal ascospore with an equatorial or subequatorial ledge. The name Saccharomycopsis praedatoria sp. nov. is proposed to accommodate the novel species. The holotype of Saccharomycopsis praedatoria is CBS 16589T. The MycoBank number is MB849369. S. praedatoria was able to kill cells of Saccharomyces cerevisiae by means of penetration with infection pegs, a trait common to most species of Saccharomycopsis.

Comparative chemical genomic profiling across plant-based hydrolysate toxins reveals widespread antagonism in fitness contributions.

The budding yeast Saccharomyces cerevisiae has been used extensively in fermentative industrial processes, including biofuel production from sustainable plant-based hydrolysates. Myriad toxins and stressors found in hydrolysates inhibit microbial metabolism and product formation. Overcoming these stresses requires mitigation strategies that include strain engineering. To identify shared and divergent mechanisms of toxicity and to implicate gene targets for genetic engineering, we used a chemical genomic approach to study fitness effects across a library of S. cerevisiae deletion mutants cultured anaerobically in dozens of individual compounds found in different types of hydrolysates. Relationships in chemical genomic profiles identified classes of toxins that provoked similar cellular responses, spanning inhibitor relationships that were not expected from chemical classification. Our results also revealed widespread antagonistic effects across inhibitors, such that the same gene deletions were beneficial for surviving some toxins but detrimental for others. This work presents a rich dataset relating gene function to chemical compounds, which both expands our understanding of plant-based hydrolysates and provides a useful resource to identify engineering targets.

Rewired cellular signaling coordinates sugar and hypoxic responses for anaerobic xylose fermentation in yeast.

Microbes can be metabolically engineered to produce biofuels and biochemicals, but rerouting metabolic flux toward products is a major hurdle without a systems-level understanding of how cellular flux is controlled. To understand flux rerouting, we investigated a panel of Saccharomyces cerevisiae strains with progressive improvements in anaerobic fermentation of xylose, a sugar abundant in sustainable plant biomass used for biofuel production. We combined comparative transcriptomics, proteomics, and phosphoproteomics with network analysis to understand the physiology of improved anaerobic xylose fermentation. Our results show that upstream regulatory changes produce a suite of physiological effects that collectively impact the phenotype. Evolved strains show an unusual co-activation of Protein Kinase A (PKA) and Snf1, thus combining responses seen during feast on glucose and famine on non-preferred sugars. Surprisingly, these regulatory changes were required to mount the hypoxic response when cells were grown on xylose, revealing a previously unknown connection between sugar source and anaerobic response. Network analysis identified several downstream transcription factors that play a significant, but on their own minor, role in anaerobic xylose fermentation, consistent with the combinatorial effects of small-impact changes. We also discovered that different routes of PKA activation produce distinct phenotypes: deletion of the RAS/PKA inhibitor IRA2 promotes xylose growth and metabolism, whereas deletion of PKA inhibitor BCY1 decouples growth from metabolism to enable robust fermentation without division. Comparing phosphoproteomic changes across ira2Δ and bcy1Δ strains implicated regulatory changes linked to xylose-dependent growth versus metabolism. Together, our results present a picture of the metabolic logic behind anaerobic xylose flux and suggest that widespread cellular remodeling, rather than individual metabolic changes, is an important goal for metabolic engineering.

Crabtree/Warburg-like aerobic xylose fermentation by engineered Saccharomyces cerevisiae.

Bottlenecks in the efficient conversion of xylose into cost-effective biofuels have limited the widespread use of plant lignocellulose as a renewable feedstock. The yeast Saccharomyces cerevisiae ferments glucose into ethanol with such high metabolic flux that it ferments high concentrations of glucose aerobically, a trait called the Crabtree/Warburg Effect. In contrast to glucose, most engineered S. cerevisiae strains do not ferment xylose at economically viable rates and yields, and they require respiration to achieve sufficient xylose metabolic flux and energy return for growth aerobically. Here, we evolved respiration-deficient S. cerevisiae strains that can grow on and ferment xylose to ethanol aerobically, a trait analogous to the Crabtree/Warburg Effect for glucose. Through genome sequence comparisons and directed engineering, we determined that duplications of genes encoding engineered xylose metabolism enzymes, as well as TKL1, a gene encoding a transketolase in the pentose phosphate pathway, were the causative genetic changes for the evolved phenotype. Reengineered duplications of these enzymes, in combination with deletion mutations in HOG1, ISU1, GRE3, and IRA2, increased the rates of aerobic and anaerobic xylose fermentation. Importantly, we found that these genetic modifications function in another genetic background and increase the rate and yield of xylose-to-ethanol conversion in industrially relevant switchgrass hydrolysate, indicating that these specific genetic modifications may enable the sustainable production of industrial biofuels from yeast. We propose a model for how key regulatory mutations prime yeast for aerobic xylose fermentation by lowering the threshold for overflow metabolism, allowing mutations to increase xylose flux and to redirect it into fermentation products.

Correction: Directed Evolution Reveals Unexpected Epistatic Interactions That Alter Metabolic Regulation and Enable Anaerobic Xylose Use by Saccharomyces cerevisiae.

[This corrects the article DOI: 10.1371/journal.pgen.1006372.].

Directed Evolution Reveals Unexpected Epistatic Interactions That Alter Metabolic Regulation and Enable Anaerobic Xylose Use by Saccharomyces cerevisiae.

The inability of native Saccharomyces cerevisiae to convert xylose from plant biomass into biofuels remains a major challenge for the production of renewable bioenergy. Despite extensive knowledge of the regulatory networks controlling carbon metabolism in yeast, little is known about how to reprogram S. cerevisiae to ferment xylose at rates comparable to glucose. Here we combined genome sequencing, proteomic profiling, and metabolomic analyses to identify and characterize the responsible mutations in a series of evolved strains capable of metabolizing xylose aerobically or anaerobically. We report that rapid xylose conversion by engineered and evolved S. cerevisiae strains depends upon epistatic interactions among genes encoding a xylose reductase (GRE3), a component of MAP Kinase (MAPK) signaling (HOG1), a regulator of Protein Kinase A (PKA) signaling (IRA2), and a scaffolding protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis (ISU1). Interestingly, the mutation in IRA2 only impacted anaerobic xylose consumption and required the loss of ISU1 function, indicating a previously unknown connection between PKA signaling, Fe-S cluster biogenesis, and anaerobiosis. Proteomic and metabolomic comparisons revealed that the xylose-metabolizing mutant strains exhibit altered metabolic pathways relative to the parental strain when grown in xylose. Further analyses revealed that interacting mutations in HOG1 and ISU1 unexpectedly elevated mitochondrial respiratory proteins and enabled rapid aerobic respiration of xylose and other non-fermentable carbon substrates. Our findings suggest a surprising connection between Fe-S cluster biogenesis and signaling that facilitates aerobic respiration and anaerobic fermentation of xylose, underscoring how much remains unknown about the eukaryotic signaling systems that regulate carbon metabolism.

Ubiquitin ligase Siah2 regulates RevErbα degradation and the mammalian circadian clock.

Regulated degradation of proteins by the proteasome is often critical to their function in dynamic cellular pathways. The molecular clock underlying mammalian circadian rhythms relies on the rhythmic expression and degradation of its core components. However, because the tools available for identifying the mechanisms underlying the degradation of a specific protein are limited, the mechanisms regulating clock protein degradation are only beginning to be elucidated. Here we describe a cell-based functional screening approach designed to quickly identify the ubiquitin E3 ligases that induce the degradation of potentially any protein of interest. We screened the nuclear hormone receptor RevErbα (Nr1d1), a key constituent of the mammalian circadian clock, for E3 ligases that regulate its stability and found Seven in absentia2 (Siah2) to be a key regulator of RevErbα stability. Previously implicated in hypoxia signaling, Siah2 overexpression destabilizes RevErbα/ß, and siRNA depletion of Siah2 stabilizes endogenous RevErbα. Moreover, Siah2 depletion delays circadian degradation of RevErbα and lengthens period length. These results demonstrate the utility of functional screening approaches for identifying regulators of protein stability and reveal Siah2 as a previously unidentified circadian clockwork regulator that mediates circadian RevErbα turnover.

Machine learning helps identify CHRONO as a circadian clock component.

Over the last decades, researchers have characterized a set of "clock genes" that drive daily rhythms in physiology and behavior. This arduous work has yielded results with far-reaching consequences in metabolic, psychiatric, and neoplastic disorders. Recent attempts to expand our understanding of circadian regulation have moved beyond the mutagenesis screens that identified the first clock components, employing higher throughput genomic and proteomic techniques. In order to further accelerate clock gene discovery, we utilized a computer-assisted approach to identify and prioritize candidate clock components. We used a simple form of probabilistic machine learning to integrate biologically relevant, genome-scale data and ranked genes on their similarity to known clock components. We then used a secondary experimental screen to characterize the top candidates. We found that several physically interact with known clock components in a mammalian two-hybrid screen and modulate in vitro cellular rhythms in an immortalized mouse fibroblast line (NIH 3T3). One candidate, Gene Model 129, interacts with BMAL1 and functionally represses the key driver of molecular rhythms, the BMAL1/CLOCK transcriptional complex. Given these results, we have renamed the gene CHRONO (computationally highlighted repressor of the network oscillator). Bi-molecular fluorescence complementation and co-immunoprecipitation demonstrate that CHRONO represses by abrogating the binding of BMAL1 to its transcriptional co-activator CBP. Most importantly, CHRONO knockout mice display a prolonged free-running circadian period similar to, or more drastic than, six other clock components. We conclude that CHRONO is a functional clock component providing a new layer of control on circadian molecular dynamics.

ESRP1 and ESRP2 are epithelial cell-type-specific regulators of FGFR2 splicing.

Cell-type-specific expression of epithelial and mesenchymal isoforms of Fibroblast Growth Factor Receptor 2 (FGFR2) is achieved through tight regulation of mutually exclusive exons IIIb and IIIc, respectively. Using an application of cell-based cDNA expression screening, we identified two paralogous epithelial cell-type-specific RNA-binding proteins that are essential regulators of FGFR2 splicing. Ectopic expression of either protein in cells that express FGFR2-IIIc caused a switch in endogenous FGFR2 splicing to the epithelial isoform. Conversely, knockdown of both factors in cells that express FGFR2-IIIb by RNA interference caused a switch from the epithelial to mesenchymal isoform. These factors also regulate splicing of CD44, p120-Catenin (CTNND1), and hMena (ENAH), three transcripts that undergo changes in splicing during the epithelial-to-mesenchymal transition (EMT). These studies suggest that Epithelial Splicing Regulatory Proteins 1 and 2 (ESRP1 and ESRP2) are coordinators of an epithelial cell-type-specific splicing program.

Mechanism of imidazolium ionic liquids toxicity in Saccharomyces cerevisiae and rational engineering of a tolerant, xylose-fermenting strain.

BACKGROUND: Imidazolium ionic liquids (IILs) underpin promising technologies that generate fermentable sugars from lignocellulose for future biorefineries. However, residual IILs are toxic to fermentative microbes such as Saccharomyces cerevisiae, making IIL-tolerance a key property for strain engineering. To enable rational engineering, we used chemical genomic profiling to understand the effects of IILs on S. cerevisiae. RESULTS: We found that IILs likely target mitochondria as their chemical genomic profiles closely resembled that of the mitochondrial membrane disrupting agent valinomycin. Further, several deletions of genes encoding mitochondrial proteins exhibited increased sensitivity to IIL. High-throughput chemical proteomics confirmed effects of IILs on mitochondrial protein levels. IILs induced abnormal mitochondrial morphology, as well as altered polarization of mitochondrial membrane potential similar to valinomycin. Deletion of the putative serine/threonine kinase PTK2 thought to activate the plasma-membrane proton efflux pump Pma1p conferred a significant IIL-fitness advantage. Conversely, overexpression of PMA1 conferred sensitivity to IILs, suggesting that hydrogen ion efflux may be coupled to influx of the toxic imidazolium cation. PTK2 deletion conferred resistance to multiple IILs, including [EMIM]Cl, [BMIM]Cl, and [EMIM]Ac. An engineered, xylose-converting ptk2∆ S. cerevisiae (Y133-IIL) strain consumed glucose and xylose faster and produced more ethanol in the presence of 1 % [BMIM]Cl than the wild-type PTK2 strain. We propose a model of IIL toxicity and resistance. CONCLUSIONS: This work demonstrates the utility of chemical genomics-guided biodesign for development of superior microbial biocatalysts for the ever-changing landscape of fermentation inhibitors.

Feedback repression is required for mammalian circadian clock function.

Direct evidence for the requirement of transcriptional feedback repression in circadian clock function has been elusive. Here, we developed a molecular genetic screen in mammalian cells to identify mutants of the circadian transcriptional activators CLOCK and BMAL1, which were uncoupled from CRYPTOCHROME (CRY)-mediated transcriptional repression. Notably, mutations in the PER-ARNT-SIM domain of CLOCK and the C terminus of BMAL1 resulted in synergistic insensitivity through reduced physical interactions with CRY. Coexpression of these mutant proteins in cultured fibroblasts caused arrhythmic phenotypes in population and single-cell assays. These data demonstrate that CRY-mediated repression of the CLOCK/BMAL1 complex activity is required for maintenance of circadian rhythmicity and provide formal proof that transcriptional feedback is required for mammalian clock function.

Harnessing genetic diversity in Saccharomyces cerevisiae for fermentation of xylose in hydrolysates of alkaline hydrogen peroxide-pretreated biomass.

The fermentation of lignocellulose-derived sugars, particularly xylose, into ethanol by the yeast Saccharomyces cerevisiae is known to be inhibited by compounds produced during feedstock pretreatment. We devised a strategy that combined chemical profiling of pretreated feedstocks, high-throughput phenotyping of genetically diverse S. cerevisiae strains isolated from a range of ecological niches, and directed engineering and evolution against identified inhibitors to produce strains with improved fermentation properties. We identified and quantified for the first time the major inhibitory compounds in alkaline hydrogen peroxide (AHP)-pretreated lignocellulosic hydrolysates, including Na(+), acetate, and p-coumaric (pCA) and ferulic (FA) acids. By phenotyping these yeast strains for their abilities to grow in the presence of these AHP inhibitors, one heterozygous diploid strain tolerant to all four inhibitors was selected, engineered for xylose metabolism, and then allowed to evolve on xylose with increasing amounts of pCA and FA. After only 149 generations, one evolved isolate, GLBRCY87, exhibited faster xylose uptake rates in both laboratory media and AHP switchgrass hydrolysate than its ancestral GLBRCY73 strain and completely converted 115 g/liter of total sugars in undetoxified AHP hydrolysate into more than 40 g/liter ethanol. Strikingly, genome sequencing revealed that during the evolution from GLBRCY73, the GLBRCY87 strain acquired the conversion of heterozygous to homozygous alleles in chromosome VII and amplification of chromosome XIV. Our approach highlights that simultaneous selection on xylose and pCA or FA with a wild S. cerevisiae strain containing inherent tolerance to AHP pretreatment inhibitors has potential for rapid evolution of robust properties in lignocellulosic biofuel production.

Comparative genomics of xylose-fermenting fungi for enhanced biofuel production.

Cellulosic biomass is an abundant and underused substrate for biofuel production. The inability of many microbes to metabolize the pentose sugars abundant within hemicellulose creates specific challenges for microbial biofuel production from cellulosic material. Although engineered strains of Saccharomyces cerevisiae can use the pentose xylose, the fermentative capacity pales in comparison with glucose, limiting the economic feasibility of industrial fermentations. To better understand xylose utilization for subsequent microbial engineering, we sequenced the genomes of two xylose-fermenting, beetle-associated fungi, Spathaspora passalidarum and Candida tenuis. To identify genes involved in xylose metabolism, we applied a comparative genomic approach across 14 Ascomycete genomes, mapping phenotypes and genotypes onto the fungal phylogeny, and measured genomic expression across five Hemiascomycete species with different xylose-consumption phenotypes. This approach implicated many genes and processes involved in xylose assimilation. Several of these genes significantly improved xylose utilization when engineered into S. cerevisiae, demonstrating the power of comparative methods in rapidly identifying genes for biomass conversion while reflecting on fungal ecology.

Oxygenation influences xylose fermentation and gene expression in the yeast genera Spathaspora and Scheffersomyces.

BACKGROUND: Cost-effective production of biofuels from lignocellulose requires the fermentation of D-xylose. Many yeast species within and closely related to the genera Spathaspora and Scheffersomyces (both of the order Serinales) natively assimilate and ferment xylose. Other species consume xylose inefficiently, leading to extracellular accumulation of xylitol. Xylitol excretion is thought to be due to the different cofactor requirements of the first two steps of xylose metabolism. Xylose reductase (XR) generally uses NADPH to reduce xylose to xylitol, while xylitol dehydrogenase (XDH) generally uses NAD+ to oxidize xylitol to xylulose, creating an imbalanced redox pathway. This imbalance is thought to be particularly consequential in hypoxic or anoxic environments. RESULTS: We screened the growth of xylose-fermenting yeast species in high and moderate aeration and identified both ethanol producers and xylitol producers. Selected species were further characterized for their XR and XDH cofactor preferences by enzyme assays and gene expression patterns by RNA-Seq. Our data revealed that xylose metabolism is more redox balanced in some species, but it is strongly affected by oxygen levels. Under high aeration, most species switched from ethanol production to xylitol accumulation, despite the availability of ample oxygen to accept electrons from NADH. This switch was followed by decreases in enzyme activity and the expression of genes related to xylose metabolism, suggesting that bottlenecks in xylose fermentation are not always due to cofactor preferences. Finally, we expressed XYL genes from multiple Scheffersomyces species in a strain of Saccharomyces cerevisiae. Recombinant S. cerevisiae expressing XYL1 from Scheffersomyces xylosifermentans, which encodes an XR without a cofactor preference, showed improved anaerobic growth on xylose as the primary carbon source compared to S. cerevisiae strain expressing XYL genes from Scheffersomyces stipitis. CONCLUSION: Collectively, our data do not support the hypothesis that xylitol accumulation occurs primarily due to differences in cofactor preferences between xylose reductase and xylitol dehydrogenase; instead, gene expression plays a major role in response to oxygen levels. We have also identified the yeast Sc. xylosifermentans as a potential source for genes that can be engineered into S. cerevisiae to improve xylose fermentation and biofuel production.

A role for ion homeostasis in yeast ionic liquid tolerance.

The model yeast Saccharomyces cerevisiae is being developed as a biocatalyst for the conversion of renewable lignocellulosic biomass into biofuels. The ionic liquid 1-ethyl-3-methylimidazolium chloride (EMIMCl) solubilizes lignocellulose for deconstruction into fermentable sugars, but it inhibits yeast fermentation. EMIMCl tolerance is mediated by the efflux pump Sge1p and uncharacterized protein Ilt1p. Through genetic investigation, we found that disruption of ion homeostasis through mutations in genes encoding the Trk1p potassium transporter and its protein kinase regulators, Sat4p and Hal5p, causes EMIMCl sensitivity. These results suggest that maintenance of ion homeostasis is important for tolerance to EMIMCl.

Transcriptomic Data Sets for Zymomonas mobilis 2032 during Fermentation of Ammonia Fiber Expansion (AFEX)-Pretreated Corn Stover and Switchgrass Hydrolysates.

The transcriptomes of Zymomonas mobilis 2032 were captured during the fermentation of ammonia fiber expansion (AFEX)-pretreated corn stover and switchgrass hydrolysates containing different concentrations of glucose and xylose. RNA samples were collected when Z. mobilis was fermenting glucose or xylose. Here, we present transcriptome sequencing (RNA-Seq) data obtained during separate phases of glucose or xylose consumption.

Water-soluble saponins accumulate in drought-stressed switchgrass and may inhibit yeast growth during bioethanol production.

BACKGROUND: Developing economically viable pathways to produce renewable energy has become an important research theme in recent years. Lignocellulosic biomass is a promising feedstock that can be converted into second-generation biofuels and bioproducts. Global warming has adversely affected climate change causing many environmental changes that have impacted earth surface temperature and rainfall patterns. Recent research has shown that environmental growth conditions altered the composition of drought-stressed switchgrass and directly influenced the extent of biomass conversion to fuels by completely inhibiting yeast growth during fermentation. Our goal in this project was to find a way to overcome the microbial inhibition and characterize specific compounds that led to this inhibition. Additionally, we also determined if these microbial inhibitors were plant-generated compounds, by-products of the pretreatment process, or a combination of both. RESULTS: Switchgrass harvested in drought (2012) and non-drought (2010) years were pretreated using Ammonia Fiber Expansion (AFEX). Untreated and AFEX processed samples were then extracted using solvents (i.e., water, ethanol, and ethyl acetate) to selectively remove potential inhibitory compounds and determine whether pretreatment affects the inhibition. High solids loading enzymatic hydrolysis was performed on all samples, followed by fermentation using engineered Saccharomyces cerevisiae. Fermentation rate, cell growth, sugar consumption, and ethanol production were used to evaluate fermentation performance. We found that water extraction of drought-year switchgrass before AFEX pretreatment reduced the inhibition of yeast fermentation. The extracts were analyzed using liquid chromatography-mass spectrometry (LC-MS) to detect compounds enriched in the extracted fractions. Saponins, a class of plant-generated triterpene or steroidal glycosides, were found to be significantly more abundant in the water extracts from drought-year (inhibitory) switchgrass. The inhibitory nature of the saponins in switchgrass hydrolysate was validated by spiking commercially available saponin standard (protodioscin) in non-inhibitory switchgrass hydrolysate harvested in normal year. CONCLUSIONS: Adding a water extraction step prior to AFEX-pretreatment of drought-stressed switchgrass effectively overcame inhibition of yeast growth during bioethanol production. Saponins appear to be generated by the plant as a response to drought as they were significantly more abundant in the drought-stressed switchgrass water extracts and may contribute toward yeast inhibition in drought-stressed switchgrass hydrolysates.

Comparative functional genomics identifies an iron-limited bottleneck in a Saccharomyces cerevisiae strain with a cytosolic-localized isobutanol pathway.

Metabolic engineering strategies have been successfully implemented to improve the production of isobutanol, a next-generation biofuel, in Saccharomyces cerevisiae. Here, we explore how two of these strategies, pathway re-localization and redox cofactor-balancing, affect the performance and physiology of isobutanol producing strains. We equipped yeast with isobutanol cassettes which had either a mitochondrial or cytosolic localized isobutanol pathway and used either a redox-imbalanced (NADPH-dependent) or redox-balanced (NADH-dependent) ketol-acid reductoisomerase enzyme. We then conducted transcriptomic, proteomic and metabolomic analyses to elucidate molecular differences between the engineered strains. Pathway localization had a large effect on isobutanol production with the strain expressing the mitochondrial-localized enzymes producing 3.8-fold more isobutanol than strains expressing the cytosolic enzymes. Cofactor-balancing did not improve isobutanol titers and instead the strain with the redox-imbalanced pathway produced 1.5-fold more isobutanol than the balanced version, albeit at low overall pathway flux. Functional genomic analyses suggested that the poor performances of the cytosolic pathway strains were in part due to a shortage in cytosolic Fe-S clusters, which are required cofactors for the dihydroxyacid dehydratase enzyme. We then demonstrated that this cofactor limitation may be partially recovered by disrupting iron homeostasis with a fra2 mutation, thereby increasing cellular iron levels. The resulting isobutanol titer of the fra2 null strain harboring a cytosolic-localized isobutanol pathway outperformed the strain with the mitochondrial-localized pathway by 1.3-fold, demonstrating that both localizations can support flux to isobutanol.