Tremendous plant metabolic diversity arises from phylogenetically restricted specialized metabolic pathways. Specialized metabolites are synthesized in dedicated cells or tissues, with pathway genes sometimes colocalizing in biosynthetic gene clusters (BGCs). However, the mechanisms by which spatial expression patterns arise and the role of BGCs in pathway evolution remain underappreciated. In this study, we investigated the mechanisms driving acylsugar evolution in the Solanaceae. Previously thought to be restricted to glandular trichomes, acylsugars were recently found in cultivated tomato roots. We demonstrated that acylsugars in cultivated tomato roots and trichomes have different sugar cores, identified root-enriched paralogs of trichome acylsugar pathway genes, and characterized a key paralog required for root acylsugar biosynthesis, SlASAT1-LIKE ( SlASAT1-L ), which is nested within a previously reported trichome acylsugar BGC. Last, we provided evidence that ASAT1-L arose through duplication of its paralog, ASAT1 , and was trichome-expressed before acquiring root-specific expression in the Solanum genus. Our results illuminate the genomic context and molecular mechanisms underpinning metabolic diversity in plants.

Solanaceae (nightshade family) species synthesize a remarkable array of clade- and tissue-specific specialized metabolites. Protective acylsugars, one such class of structurally diverse metabolites, are produced by ACYLSUGAR ACYLTRANSFERASE (ASAT) enzymes from sugars and acyl-coenzyme A esters. Published research has revealed trichome acylsugars composed of glucose and sucrose cores in species across the family. In addition, acylsugars have been analyzed across a small fraction of the >1,200 species in the phenotypically megadiverse Solanum genus, with a handful containing inositol and glycosylated inositol cores. The current study sampled several dozen species across subclades of Solanum to get a more detailed view of acylsugar chemodiversity. In depth characterization of acylsugars from the clade II species brinjal eggplant (Solanum melongena) led to the identification of eight unusual structures with inositol or inositol glycoside cores and hydroxyacyl chains. Liquid chromatography-mass spectrometry analysis of 31 additional species in the Solanum genus revealed striking acylsugar diversity, with some traits restricted to specific clades and species. Acylinositols and inositol-based acyldisaccharides were detected throughout much of the genus. In contrast, acylglucoses and acylsucroses were more restricted in distribution. Analysis of tissue-specific transcriptomes and interspecific acylsugar acetylation differences led to the identification of the brinjal eggplant ASAT 3-LIKE 1 (SmASAT3-L1; SMEL4.1_12g015780) enzyme. This enzyme is distinct from previously characterized acylsugar acetyltransferases, which are in the ASAT4 clade, and appears to be a functionally divergent ASAT3. This study provides a foundation for investigating the evolution and function of diverse Solanum acylsugar structures and harnessing this diversity in breeding and synthetic biology.

Introduction Larvae of the invasive emerald ash borer [EAB, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae)], kill over 99% of green ash ( Fraxinus pennsylvanica Marshall) trees they infest, yet a small percentage of green ash (“lingering ash”) survive years of heavy EAB attack. In the face of an ongoing invasion that threatens multiple North American Fraxinus species with extinction, any evidence for reproducible defensive responses in the native species merits investigation. Methods We evaluated the capacity of three families of green ash F 1 progeny to kill EAB larvae when challenged in greenhouse studies by infestation with a uniform density of EAB eggs followed by dissection 8 weeks post-infestation and comparison of the host metabolomic profiles. Results The mean proportions of host-killed larvae in the two families of F 1 progeny from lingering ash parents were significantly higher than that of host-killed larvae in the family of F 1 progeny from susceptible ash parents ( p < 0.001). Untargeted metabolomics comparing F 1 progeny in the quartile with the highest percent host-killed larvae (HHK) to F 1 progeny in the quartile with the lowest percent host-killed larvae (LHK) and to the uninfested F 1 progeny within each family revealed evidence for induced biochemical responses to EAB. Infested trees produced significantly higher levels of select secoiridoids than uninfested trees, and LHK progeny produced significantly higher levels of select secoiridoids than the HHK progeny. HHK progeny produced significantly higher abundances of three metabolites annotated as aromatic alkaloids than the LHK and uninfested individuals. Discussion Based on these results, we hypothesize that green ash responds to EAB infestation. However, only certain trees have the genetic capacity to tailor a response that kills enough EAB larvae to prevent lethal damage to the vascular system. Rigorous tests of this hypothesis will require 15–20 years of additional crossing, phenotyping, and omics analyses. The results of this investigation will encourage the establishment and continuation of breeding programs that, in concert with biocontrol and management, could provide trees that slow, if not halt, the decimation of the Fraxinus gene pool. At the same time, ongoing work on host-insect interaction will contribute to our understanding of how forest trees recognize and defend themselves against phloem-feeding insects.

Methods for collection of placental tissue at room temperature for metabolic profiling are described. Specimens were excised from the maternal side of the placenta and immediately flash frozen or fixed and stored for 1, 6, 12, 24, or 48 h in 80% methanol. Untargeted metabolic profiling was performed on both the methanol-fixed tissue and the methanol extract. Data were analyzed using Gaussian generalized estimating equations, two sample t-tests with false discovery rate (FDR) corrections, and principal components analysis. Methanol-fixed tissue samples and methanol extracts had a similar number of metabolites (p = 0.45, p = 0.21 in positive vs. negative ion mode). In positive ion mode, when compared to flash frozen tissue, both the methanol extract and methanol-fixed tissue (6 h) had a higher number of metabolites detected (146 additional metabolites, p FDR = 0.020; 149 additional metabolites, p FDR = 0.017; respectively), but these associations were not found in negative ion mode (all p FDR ≥ 0.05). Principle components analysis demonstrated separation of the metabolite features in the methanol extract, but similarity between methanol-fixed tissue and flash frozen tissue. These results show that placental tissue samples collected in 80% methanol at room temperature can yield similar metabolic data to flash frozen specimens.

The role of metabolites, nutrients, and toxins (MNTs) in sera at the end of pregnancy and of their association with offspring respiratory and allergic disorders is underexplored. Untargeted approaches detecting a variety of compounds, known and unknown, are limited. In this cohort study, we first aimed at discovering associations of MNTs in grandmaternal (F0) serum with asthma, immunoglobulin E, skin prick tests, exhaled nitric oxide, and lung function parameters in their parental (F1) offspring. Second, for replication, we tested the identified associations of MNTs with disorders in their grandchildren (F2-offspring) based on F2 cord serum. The statistical analyses were sex-stratified. Using liquid chromatography/high-resolution mass spectrometry in F0, we detected signals for 2286 negative-ion lipids, 59 positive-ion lipids, and 6331 polar MNTs. Nine MNTs (one unknown MNT) discovered in F0-F1 and replicated in F2 showed higher risks of respiratory/allergic outcomes. Twelve MNTs (four unknowns) constituted a potential protection in F1 and F2. We recognized MNTs not yet considered candidates for respiratory/allergic outcomes: a phthalate plasticizer, an antihistamine, a bile acid metabolite, tryptophan metabolites, a hemiterpenoid glycoside, triacylglycerols, hypoxanthine, and polyphenol syringic acid. The findings suggest that MNTs are aspirants for clinical trials to prevent adverse respiratory/allergic outcomes.

Maternal smoking in pregnancy (MSP) affects the offspring's DNA methylation (DNAm). There is a lack of knowledge regarding individual differences in susceptibility to exposure to MSP. Glutathione S-transferase (GST) genes are involved in protection against harmful oxidants such as those found in cigarette smoke. This study aimed to test whether polymorphisms in GST genes influence the effect of MSP on offspring DNAm. Using data from the Isle of Wight birth cohort, we assessed the association of MSP and offspring DNAm in 493 mother-child dyads (251 male, 242 female) with the effect-modifying role of GST gene polymorphism (at rs506008, rs574344, rs12736389, rs3768490, rs1537234, and rs1695). MSP was assessed by levels of nicotine and its downstream metabolites (cotinine, norcotinine, and hydroxycotinine) in maternal sera. In males, associations of hydroxycotinine with DNAm at cg18473733, cg25949550, cg11647108, and cg01952185 and norcotinine with DNAm at cg09935388 were modified by GST gene polymorphisms (p-values < 0.05). In females, associations of hydroxycotinine with DNAm at cg12160087 and norcotinine with DNAm at cg18473733 were modified by GST gene polymorphisms (p-values < 0.05). Our study emphasizes the role of genetic polymorphism in GST genes in DNAm's susceptibility to MSP.

Fucoxanthin, a marine xanthophyll carotenoid, has been shown to exert beneficial health effects. Cell‐based and animal‐based experimental studies have shown that fucoxanthin has the potential to mitigate eczema symptoms. Hence, we sought to assess whether fucoxanthinol 3‐arachidate, a fucoxanthin metabolite, measured in maternal serum at birth is associated with eczema development during early childhood.

Summary Plant‐specialized metabolism is complex, with frequent examples of highly branched biosynthetic pathways, and shared chemical intermediates. As such, many plant‐specialized metabolic networks are poorly characterized. The N‐methyl Δ1‐pyrrolinium cation is a simple pyrrolidine alkaloid and precursor of pharmacologically important tropane alkaloids. Silencing of pyrrolidine ketide synthase (AbPyKS) in the roots of Atropa belladonna (Deadly Nightshade) reduces tropane alkaloid abundance and causes high N‐methyl Δ1‐pyrrolinium cation accumulation. The consequences of this metabolic shift on alkaloid metabolism are unknown. In this study, we utilized discovery metabolomics coupled with AbPyKS silencing to reveal major changes in the root alkaloid metabolome of A. belladonna. We discovered and annotated almost 40 pyrrolidine alkaloids that increase when AbPyKS activity is reduced. Suppression of phenyllactate biosynthesis, combined with metabolic engineering in planta, and chemical synthesis indicates several of these pyrrolidines share a core structure formed through the nonenzymatic Mannich‐like decarboxylative condensation of the N‐methyl Δ1‐pyrrolinium cation with 2‐O‐malonylphenyllactate. Decoration of this core scaffold through hydroxylation and glycosylation leads to mono‐ and dipyrrolidine alkaloid diversity. This study reveals the previously unknown complexity of the A. belladonna root metabolome and creates a foundation for future investigation into the biosynthesis, function, and potential utility of these novel alkaloids.

The emerald ash borer ( Agrilus planipennis , EAB) invasion in North America threatens most North American Fraxinus species, including green ash ( F. pennsylvanica ), the mostly widely distributed species (1, 2). A small number of green ash (“lingering ash”, 0.1-1%) survive years of heavy EAB attack (3) and kill more EAB larvae when challenged in greenhouse studies than susceptible controls (4). We combined untargeted metabolomics with intensive phenotyping of segregating F 1 progeny from susceptible or lingering ash parents to detect chemotypes associated with defensive responses to EAB. We examined three contrasting groups: low larval kill (0-25% of larvae killed), high larval kill (55-95% of larvae killed) and uninfested. Contrasting the chemotypes of these groups revealed evidence of an induced response to EAB. Infested trees deployed significantly higher levels of select secoiridoids than uninfested trees. Within the infested group, the low larval kill (LLK) individuals deployed significantly higher levels of select secoiridoids than the high larval kill (HLK) individuals. The HLK individuals deployed significantly higher concentrations of three metabolites annotated as aromatic alkaloids compared to the LLK and uninfested individuals. We propose a two-part model for the North American Fraxinus response to EAB wherein every individual has the capacity to detect and respond to EAB, but only certain trees mount an effective defense, killing enough EAB larvae to prevent or minimize lethal damage to the vascular system. Integration of intensive phenotyping of structured populations with metabolomics reveals the multi-faceted nature of the defenses deployed in naïve host populations against invasive species. Significance Long-lived forest trees employ evolutionarily conserved templates to synthesize an array of defensive metabolites. The regulation of these metabolites, honed against native pests and pathogens, may be ineffective against novel species, as illustrated by the high mortality (>99%) in green ash infested by the invasive emerald ash borer (EAB). However, high standing genetic variation may produce a few individuals capable of an effective defense, as seen in the rare surviving green ash. In an investigation of this plant-insect interaction, we annotated metabolites associated with generalized but ineffective responses to EAB, and others associated with successful defensive responses. Untargeted metabolomics combined with intensive phenotyping of structured populations provides a framework for understanding resistance to invasive species in naïve host populations.

Bile acids (BAs) are steroid detergents in bile that contribute to fat absorption, cell signaling, and microbiome interactions. The final step in their synthesis is amino acid conjugation with either glycine or taurine in the liver by the enzyme bile acid-CoA:amino acid N-acyltransferase (BAAT). Here, we describe the microbial, chemical, and physiological consequences of Baat gene knockout. Baat-/- mice were underweight after weaning but quickly exhibited catch-up growth. At three weeks of age, KO animals had increased phospholipid excretion and decreased subcutaneous fat pad mass, liver mass, glycogen staining in hepatocytes, and hepatic vitamin A stores, but these were less marked in adulthood. Additionally, KO mice had an altered microbiome in early life. Their BA pool was highly enriched in cholic acid but not completely devoid of conjugated BAs. KO animals had 27-fold lower taurine-conjugated BAs than wild type in their liver but similar concentrations of glycine-conjugated BAs and higher microbially conjugated BAs. Furthermore, the BA pool in Baat-/- was enriched in a variety of unusual BAs that were putatively sourced from cysteamine conjugation with subsequent oxidation and methylation of the sulfur group mimicking taurine. Antibiotic treatment of KO mice indicated the microbiome was not the likely source of the unusual conjugations, instead, the unique BAs in KO animals were likely derived from the peroxisomal acyltransferases Acnat1 and Acnat2, which are duplications of Baat in the mouse genome that are inactivated in humans. This study demonstrates that BA conjugation is important for early life development of mice.

Lignin-carbohydrate complexes (LCCs) in the plant cell wall are responsible for providing resistance against biomass-degrading enzymes produced by microorganisms. Four major types of lignin-carbohydrate bonds are reported in the literature, namely, benzyl ethers, benzyl esters, phenyl glycosides, and acetyl ester linkages. Ester's linkages in the plant cell wall are labile to alkaline pretreatments, such as ammonia fiber expansion (AFEX), which uses liquid or gaseous ammonia to cleave those linkages in the plant cell wall and reduce biomass recalcitrance. Two competing reactions, notably hydrolysis and ammonolysis, take place during AFEX pretreatment process, producing different aliphatic and aromatic acids, as well as their amide counterparts. AFEX pretreated grasses and agricultural residues are known to increase conversion of biomass to sugars by four- to five-fold when subjected to commercial enzyme hydrolysis, yielding a sustainable feedstock for producing biofuels, biomaterials, and animal feed. Animal feed trials on dairy cows have demonstrated a 27% increase in milk production when compared to a control feedstock. However, the presence of carboxamides in feedstocks could promote neurotoxicity in animals if consumed beyond a certain concentration. Thus, there is the need to overcome regulatory hurdles associated with commercializing AFEX pretreated biomass as animal feed in the United States. This manuscript demonstrates a modified pretreatment for increasing the digestibility of industrial byproducts such as Brewer's spent grains (BSG) and high-fiber meal (HFM) produced from BSG and dry distillers grains with soluble (DDGS), while avoiding the production of carboxamides. The three industrial byproducts were first treated with calculated amounts of alkali such as NaOH, Ca(OH)2, or KOH followed by AFEX pretreatment. We found that 4% alkali was able to de-esterify BSG and DDGS more efficiently than using 2% alkali at both 10 and 20% solids loading. AFEX pretreatment of de-esterified BSG, HFM, and DDGS produced twofold higher glucan conversion than respective untreated biomass. This new discovery can help overcome potential regulatory issues associated with the presence of carboxamides in ammonia-pretreated animal feeds and is expected to benefit several farmers around the world.

Identifying and quantifying mixtures of compounds with very similar fragmentation patterns in their mass spectra presents a unique and challenging problem. In particular, the mass spectra of most per- and poly-fluoroalkyl substances (PFAS) lack a molecular ion. This complicates their identification, especially when using the absence of chromatographic separation. Here, we focus on linear, nonpolar, short-chain PFAS, which have received less attention than amphipathic PFAS despite their longer environmental lifetimes and greater global warming potentials. We identify and quantify n-C5F12 and n-C6F14 in binary mixtures by analyzing small changes in abundances of the main fragment ions following femtosecond tunnel laser ionization, without the need of chromatographic separation. Time-resolved femtosecond ionization mass spectrometry reveals that the metastable cation of both compounds undergoes predissociation within 1-2 ps of ion formation, with yields of C3F7+ showing evidence of coherent vibrational dynamics. These coherent oscillations are compared to low-level ion-state calculations and supported the idea that the oscillations in the C3F7+ ion yield are due to vibrations in the C5F12+• and C6F14+• radical cations and are associated with the predissociation dynamics of the metastable molecular ion. Surprisingly, we find that the fragment ions used for quantifying the mixtures have similar fragmentation dynamics. Conversely, the odd-electron C2F4+• fragment shows different time dependence between the two compounds, yet has negligible difference in the relative ion yield between the two compounds. Our findings indicate that femtosecond laser ionization may be a useful tool for identifying and quantifying mixtures of PFAS without the need of chromatography or high-resolution mass spectrometry.

Defining the denatured state ensemble (DSE) and disordered proteins is essential to understanding folding, chaperone action, degradation, and translocation. As compared with water-soluble proteins, the DSE of membrane proteins is much less characterized. Here, we measure the DSE of the helical membrane protein GlpG of Escherichia coli (E. coli) in native-like lipid bilayers. The DSE was obtained using our steric trapping method, which couples denaturation of doubly biotinylated GlpG to binding of two streptavidin molecules. The helices and loops are probed using limited proteolysis and mass spectrometry, while the dimensions are determined using our paramagnetic biotin derivative and double electron-electron resonance spectroscopy. These data, along with our Upside simulations, identify the DSE as being highly dynamic, involving the topology changes and unfolding of some of the trans membrane (TM) helices. The DSE is expanded relative to the native state but only to 15 to 75% of the fully expanded condition. The degree of expansion depends on the local protein packing and the lipid composition. E. coli's lipid bilayer promotes the association of TM helices in the DSE and, probably in general, facilitates interhelical interactions. This tendency may be the outcome of a general lipophobic effect of proteins within the cell membranes.

Plants make a variety of specialized metabolites that can mediate interactions with animals, microbes and competitor plants. Understanding how plants synthesize these compounds enables studies of their biological roles by manipulating their synthesis in vivo as well as producing them in vitro . Acylsugars are a group of protective metabolites that accumulate in the trichomes of many Solanaceae family plants. Acylinositol biosynthesis is of interest because it appears to be restricted to a subgroup of species within the Solanum genus. Previous work characterized a triacylinositol acetyltransferase involved in acylinositol biosynthesis in the Andean fruit plant Solanum quitoense (lulo or naranjillo). We characterized three additional S. quitoense trichome expressed enzymes, and found that virus induced gene silencing of each caused changes in acylinositol accumulation. Surprisingly, the in vitro triacylinositol products of these enzymes are distinct from those that accumulate in planta . These enzymes, nonetheless, provide an opportunities to test the biological impact and properties of these triacylinositols in vitro .

Acylsugars are defensive, trichome-synthesized sugar esters produced in plants across the Solanaceae (nightshade) family. Although assembled from simple metabolites and synthesized by a relatively short core biosynthetic pathway, tremendous within- and across-species acylsugar structural variation is documented across the family. To advance our understanding of the diversity and the synthesis of acylsugars within the Nicotiana genus, trichome extracts were profiled across the genus coupled with transcriptomics-guided enzyme discovery and in vivo and in vitro analysis. Differences in the types of sugar cores, numbers of acylations, and acyl chain structures contributed to over 300 unique annotated acylsugars throughout Nicotiana . Placement of acyl chain length into a phylogenetic context revealed that an unsaturated acyl chain type was detected in a few closely-related species. A comparative transcriptomics approach identified trichome-enriched Nicotiana acuminata acylsugar biosynthetic candidate enzymes. > 25 acylsugar variants could be produced in a single enzyme assay with four acylsugar acyltransferases (NacASAT1-4) together with structurally diverse acyl-CoAs and sucrose. Liquid chromatography coupled with mass spectrometry screening of in vitro products revealed the ability of these enzymes to make acylsugars not present in Nicotiana plant extracts. In vitro acylsugar production also provided insights into acyltransferase acyl donor promiscuity and acyl acceptor specificity as well as regiospecificity of some ASATs. This study suggests that promiscuous Nicotiana acyltransferases can be used as synthetic biology tools to produce novel and potentially useful metabolites. ONE SENTENCE SUMMARY Analysis of Nicotiana glandular trichome metabolites and BAHD acyltransferases revealed diverse sucrose and glucose based acylesters.

Acetaminophen is used by nearly two-thirds of pregnant women. Although considered safe, studies have demonstrated associations between prenatal acetaminophen use and adverse health outcomes in offspring. Since DNA methylation (DNAm) at birth may act as an early indicator of later health, assessments on whether DNAm of newborns is associated with gestational acetaminophen use or its metabolites are needed. Using data from three consecutive generations of the Isle of Wight cohort (F0-grandmothers, F1-mothers, and F2-offspring) we investigated associations between acetaminophen metabolites in F0 serum at delivery with epigenome-wide DNAm in F1 (Guthrie cards) and between acetaminophen use of F1 and F2-cord-serum levels with F2 cord blood DNAm. In epigenome-wide screening, we eliminated non-informative DNAm sites followed by linear regression of informative sites. Based on repeated pregnancies, indication bias analyses tested whether acetaminophen indicated maternal diseases or has a risk in its own right. Considering that individuals with similar intake process acetaminophen differently, metabolites were clustered to distinguish metabolic exposures. Finally, metabolite clusters from F1-maternal and F2-cord sera were tested for their associations with newborn DNAm (F1 and F2). Twenty-one differential DNAm sites in cord blood were associated with reported maternal acetaminophen intake in the F2 generation. For 11 of these cytosine-phosphate-guanine (CpG) sites, an indication bias was excluded and five were replicated in F2 with metabolite clusters. In addition, metabolite clusters showed associations with 25 CpGs in the F0-F1 discovery analysis, of which five CpGs were replicated in the F2-generation. Our results suggest that prenatal acetaminophen use, measured as metabolites, may influence DNAm in newborns.

Plant alkaloids constitute an important class of bioactive chemicals with applications in medicine and agriculture. However, the knowledge gap of the diversity and biosynthesis of phytoalkaloids prevents systematic advances in biotechnology for engineered production of these high-value compounds. In particular, the identification of cytochrome P450s driving the structural diversity of phytoalkaloids has remained challenging. Here, we use a combination of reverse genetics with discovery metabolomics and multivariate statistical analysis followed by in planta transient assays to investigate alkaloid diversity and functionally characterize two candidate cytochrome P450s genes from Atropa belladonna without a priori knowledge of their functions or information regarding the identities of key pathway intermediates. This approach uncovered a largely unexplored root localized alkaloid sub-network that relies on pseudotropine as precursor. The two cytochrome P450s catalyze N-demethylation and ring-hydroxylation reactions within the early steps in the biosynthesis of diverse N-demethylated modified tropane alkaloids.

Switchgrass (Panicum virgatum L.) is a bioenergy crop that grows productively on lands not suitable for food production and is an excellent target for low-pesticide input biomass production. We hypothesize that resistance to insect pests and microbial pathogens is influenced by low-molecular-weight compounds known as specialized metabolites. We employed untargeted liquid chromatography-mass spectrometry, quantitative gas chromatography-mass spectrometry (GC-MS), and nuclear magnetic resonance spectroscopy to identify differences in switchgrass ecotype metabolomes. This analysis revealed striking differences between upland and lowland switchgrass metabolomes as well as distinct developmental profiles. Terpenoid- and polyphenol-derived specialized metabolites were identified, including steroidal saponins, di- and sesqui-terpenoids, and flavonoids. The saponins are particularly abundant in switchgrass extracts and have diverse aglycone cores and sugar moieties. We report seven structurally distinct steroidal saponin classes with unique steroidal cores and glycosylated at one or two positions. Quantitative GC-MS revealed differences in total saponin concentrations in the leaf blade, leaf sheath, stem, rhizome, and root (2.3 ± 0.10, 0.5 ± 0.01, 2.5 ± 0.5, 3.0 ± 0.7, and 0.3 ± 0.01 μg/mg of dw, respectively). The quantitative data also demonstrated that saponin concentrations are higher in roots of lowland (ranging from 3.0 to 6.6 μg/mg of dw) than in upland (from 0.9 to 1.9 μg/mg of dw) ecotype plants, suggesting ecotypic-specific biosynthesis and/or biological functions. These results enable future testing of these specialized metabolites on biotic and abiotic stress tolerance and can provide information on the development of low-input bioenergy crops.

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.