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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Lighting up yeast: overview of optogenetics in yeast and their applications to yeast biotechnology

Abstract Optogenetics is an empowering technology that uses light-responsive proteins to control biological processes. Because of its genetic tractability, abundance of genetic tools, and robust culturing conditions, Saccharomyces cerevisiae has served for many years as an ideal platform in which to study, develop, and apply a wide range of optogenetic systems. In many instances, yeast has been used as a steppingstone in which to characterize and optimize optogenetic tools to later be deployed in higher eukaryotes. More recently, however, optogenetic tools have been developed and deployed in yeast specifically for biotechnological applications, including in nonconventional yeasts. In this review, we summarize various optogenetic systems responding to different wavelengths of light that have been demonstrated in diverse yeast species. We then describe various applications of these optogenetic tools in yeast, particularly in metabolic engineering and recombinant protein production. Finally, we discuss emerging applications in yeast cybergenetics—the interfacing of yeast and computers for closed-loop controls of yeast bioprocesses—and the potential impact of optogenetics in other future biotechnological applications.

Biotechnology & Applied Microbiology↗
DOE OSTI2026

Data for Rewiring Yeast Metabolism for Producing 2,3-Butanediol and Two Downstream Applications: Techno-Economic Analysis and Life Cycle Assessment of Methyl Ethyl Ketone (MEK) and Agricultural Biostimulant Production

Rising concerns for sustainability and global climate change have driven the development of sustainable production pathways for biofuels and chemicals from lignocellulosic biomass via integrated biological and chemical processes. We constructed an engineered Saccharomyces cerevisiae capable of producing 2,3-butanediol (2,3-BDO) from glucose without accumulating ethanol and glycerol, which hinder downstream processing of 2,3-BDO, through extensive metabolic reprogramming. Specifically, we introduced heterologous 2,3-BDO biosynthetic enzymes and deleted the major isozymes of ethanol and glycerol biosynthetic enzymes. In addition, we introduced an NAD+ regenerating Pyruvate-Malate (PM) cycle and enhanced the NAD+ regenerating capability of the PM cycle to resolve the redox imbalance from the deletion of ethanol and glycerol production pathways. The resulting engineered yeast produced 109.9 g/L of 2,3-BDO with a productivity of 1.0 g/L/h and a yield of 0.36 g/g glucose in a fed-batch fermentation. We also conducted techno-economic analysis (TEA) and life cycle assessment (LCA) of the production of methyl ethyl ketone (MEK) through catalytic dehydration of 2,3-BDO. A TEA based on the experimental results indicated that the minimum product selling price (MPSP) was estimated to be $1.90/kg. Regarding cradle-to-grave LCA, 100-year global warming potential (GWP100) and fossil energy consumption (FEC) were found to be 0.37 kg CO2 eq/kg and 3.1 MJ/kg, respectively. These results demonstrated the feasibility of cost-competitive and sustainable bio-based MEK production via yeast fermentation. In addition, we explored the possibility of using the fermentation broth containing 2,3-BDO as a biostimulant inducing drought tolerance in plants. As a result, the yeast 2,3-BDO fermentation broth can induce drought tolerance in Arabidopsis thaliana without a complicated purification process.

Economics↗
DOE OSTI2024

Selected humanization of yeast U1 snRNP leads to global suppression of pre-mRNA splicing and mitochondrial dysfunction in the budding yeast

The recognition of the 5′ splice site (5′ ss) is one of the earliest steps of pre-mRNA splicing. To better understand, the mechanism and regulation of 5′ ss recognition, we selectively humanized components of the yeast U1 (yU1) snRNP to reveal the function of these components in 5′ ss recognition and splicing. We targeted U1C and Luc7, two proteins that interact with and stabilize the yU1 snRNA and the 5′ ss RNA duplex. We replaced the zinc-finger (ZnF) domain of yeast U1C (yU1C) with its human counterpart, which resulted in a cold-sensitive growth phenotype and moderate splicing defects. We next added an auxin-inducible degron to yeast Luc7 (yLuc7) protein (to mimic the lack of Luc7Ls in human U1 snRNP). We found that Luc7-depleted yU1 snRNP resulted in the concomitant loss of Prp40 and Snu71 (two other essential yU1 snRNP proteins), and further biochemical analyses suggest a model of how these three proteins interact with each other in the U1 snRNP. The loss of these proteins resulted in a significant growth retardation accompanied by a global suppression of pre-mRNA splicing. The splicing suppression led to mitochondrial dysfunction as revealed by a release of Fe 2+ into the growth medium and an induction of mitochondrial reactive oxygen species. Together, these observations indicate that the human U1C ZnF can substitute that of yeast, Luc7 is essential for the incorporation of the Luc7–Prp40–Snu71 trimer into yU1 snRNP, and splicing plays a major role in the regulation of mitochondrial function in yeast.

Biochemistry & Molecular Biology↗
DOE OSTI2026

Data for Metabolic Engineering of Nonmodel Yeast Issatchenkia orientalis SD108 for 5-Aminolevulinic Acid Production

Biological production of 5‐aminolevulinic acid (5‐ALA) has received growing attentionover theyears.However, thereis the tradeoff between 5‐ALA biosynthesis and cell growth because the fermentation broth will become acidic due to the production of 5‐ALA. To address this limitation, we engineered an acid‐tolerant yeast, Issatchenkia orientalis SD108, for 5‐ALA production. We first discovered that the cell growth rate of I. orientalis SD108 was boosted by 5‐ALA and its endogenous ALA synthetase (ALAS) showed higher activity than those homologs from other yeasts. The titer of 5‐ALA was improved from 28mg/L to 120‐, 150‐, and 300mg/L, by optimizing plasmid design, overexpressing a transporter, and increasing gene copy number, respectively. After redirecting the metabolic flux using the pyruvate decarboxylase (PDC) knockout strain (SD108ΔPDC) and culturing with urea, we increased the titer of 5‐ALA to 510mg/L, a 13‐fold enhancement, proving the importance of the newly identified IoALAS with higher activity and the strategic selection of nitrogen sources for knockout strains. This study demonstrates the acid‐tolerant I. orientalis SD108ΔPDC has a high potential for 5‐ALA production at a large scale in the future.

Bioproducts↗
DOE OSTI2026

Data for Resourceful and Economical Designing of Fermentation Medium for Lab and Commercial Strains of Yeast from Alternative Feedstock: Transgenic Oilcane

Sugarcane plant engineered to accumulate lipids in its vegetative tissue is being developed as a new bioenergy crop. The new crop would be a source of juice, oil, and cellulosic sugars. However, limited tolerance of industrially recognized yeasts towards inhibitors generated during the processing of lignocellulosic biomass to produce fermentable sugars is a major challenge in developing scalable processes for second-generation drop-in fuel production. To this end, hydrolysates generated from engineered sugarcane—‘oilcane’ bagasse contain added phenolics and fatty acids that further restrict the growth of fermenting microorganisms and necessitate nutrient supplementation and/or detoxification of hydrolysate which makes the fermentation process expensive. Herein, we propose a resourceful and economical approach for growing lab and commercial strains of S. cerevisiae on unrefined cellulosic sugars aerobically and fermentatively. An equal ratio of hydrolysate and juice was found optimum for growth and fermentation by lab and commercial strains of Saccharomyces cerevisiae engineered for xylose fermentation. The industrial strain grew and fermented efficiently under low aeration conditions having an ethanol titer, yield, specific and volumetric productivities of 46.96 ± 0.19 g/l, 0.51 ± 0.00 g/g, 0.27 ± 0.02 g/g.h and 1.95 ± 0.01 g/l.h, respectively, while the lab strain grew better under higher aeration conditions having the ethanol titer, yield, specific and volumetric productivities of 24.93 ± 0.09, 0.27 ± 0.00 g/g, 0.17 ± 0.00 g/g.h and 1.04 ± 0.00 g/l.h, respectively. Acclimation of cultures in a blended medium significantly improved the performance of the yeast strains. The addition of transgenic oilcane juice, which is inedible and rich in amino acids, to the hydrolysate averted the need for expensive nutrient supplementation and detoxification steps of hydrolysate. The approach provides an economical solution to reduce the cost of fermentation at an industrial scale for second-generation drop-in fuel production.

Biomass Analytics↗
DOE OSTI2026

Evaluating isoprenol production using the IPP-bypass pathway in the oleaginous yeast Rhodosporidium toruloides

Background To strengthen the national energy supply, there is an increasing demand for domestically generated aviation fuels. Bio-derived advanced aviation fuels offer the opportunity to meet this domestic need while presenting a unique opportunity to investigate the production of novel aviation fuels. Isoprenol, a chemical precursor to such novel fuels, has been shown to be a biologically producible compound in model organisms, but its bio-producibility needs to be further explored in organisms more compatible with industrial bioproduction. Results In this work, we evaluate isoprenol production using the promising bioproduction yeast, Rhodosporidium toruloides. First, we show successful isoprenol production using the IPP-bypass pathways most successful in laboratory strains of E. coli and S. cerevisiae. Next, we demonstrate that increased flux through the mevalonate pathway only modestly increases isoprenol titers. Using proteomics, we identified a potential bottleneck in production at the final step in the IPP-bypass pathway and explored alternative enzymes for this step. Finally, the top three strains of R. toruloides were evaluated in sorghum hydrolysates generated using cholinium lysinate. Through this work, 93.1 mg/L of isoprenol was produced in mock medium and 27.3 mg/L in sorghum hydrolysates. Conclusion Together these results lay the foundation for future work for the production of isoprenol from bioproduction crops.

Advanced aviation fuel↗
DOE OSTI2024

Saccharomycotina yeasts defy long-standing macroecological patterns

The Saccharomycotina yeasts (“yeasts” hereafter) are a fungal clade of scientific, economic, and medical significance. Yeasts are highly ecologically diverse, found across a broad range of environments in every biome and continent on earth; however, little is known about what rules govern the macroecology of yeast species and their range limits in the wild. Here, we trained machine learning models on 12,816 terrestrial occurrence records and 96 environmental variables to infer global distribution maps at ~1 km2 resolution for 186 yeast species (~15% of described species from 75% of orders) and to test environmental drivers of yeast biogeography and macroecology. We found that predicted yeast diversity hotspots occur in mixed montane forests in temperate climates. Diversity in vegetation type and topography were some of the greatest predictors of yeast species richness, suggesting that microhabitats and environmental clines are key to yeast diversity. We further found that range limits in yeasts are significantly influenced by carbon niche breadth and range overlap with other yeast species, with carbon specialists and species in high-diversity environments exhibiting reduced geographic ranges. Finally, yeasts contravene many long-standing macroecological principles, including the latitudinal diversity gradient, temperature-dependent species richness, and a positive relationship between latitude and range size (Rapoport’s rule). These results unveil how the environment governs the global diversity and distribution of species in the yeast subphylum. These high-resolution models of yeast species distributions will facilitate the prediction of economically relevant and emerging pathogenic species under current and future climate scenarios.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Hydrothermal conditioning of oleaginous yeast cells to enable recovery of lipids as potential drop-in fuel precursors

Abstract Background Lipids produced using oleaginous yeast cells are an emerging feedstock to manufacture commercially valuable oleochemicals ranging from pharmaceuticals to lipid-derived biofuels. Production of biofuels using oleaginous yeast is a multistep procedure that requires yeast cultivation and harvesting, lipid recovery, and conversion of the lipids to biofuels. The quantitative recovery of the total intracellular lipid from the yeast cells is a critical step during the development of a bioprocess. Their rigid cell walls often make them resistant to lysis. The existing methods include mechanical, chemical, biological and thermochemical lysis of yeast cell walls followed by solvent extraction. In this study, an aqueous thermal pretreatment was explored as a method for lysing the cell wall of the oleaginous yeast Rhodotorula toruloides for lipid recovery. Results Hydrothermal pretreatment for 60 min at 121 °C with a dry cell weight of 7% (w/v) in the yeast slurry led to a recovery of 84.6 ± 3.2% (w/w) of the total lipids when extracted with organic solvents. The conventional sonication and acid-assisted thermal cell lysis led to a lipid recovery yield of 99.8 ± 0.03% (w/w) and 109.5 ± 1.9% (w/w), respectively. The fatty acid profiles of the hydrothermally pretreated cells and freeze-dried control were similar, suggesting that the thermal lysis of the cells did not degrade the lipids. Conclusion This work demonstrates that hydrothermal pretreatment of yeast cell slurry at 121 °C for 60 min is a robust and sustainable method for cell conditioning to extract intracellular microbial lipids for biofuel production and provides a baseline for further scale-up and process integration. Graphical abstract

09 BIOMASS FUELS↗
DOE OSTI2024

Data for Hydrothermal Conditioning of Oleaginous Yeast Cells to Enable Recovery of Lipids as Potential Drop-in Fuel Precursors

Lipids produced using oleaginous yeast cells are an emerging feedstock to manufacture commercially valuable oleochemicals ranging from pharmaceuticals to lipid-derived biofuels. Production of biofuels using oleaginous yeast is a multistep procedure that requires yeast cultivation and harvesting, lipid recovery, and conversion of the lipids to biofuels. The quantitative recovery of the total intracellular lipid from the yeast cells is a critical step during the development of a bioprocess. Their rigid cell walls often make them resistant to lysis. The existing methods include mechanical, chemical, biological and thermochemical lysis of yeast cell walls followed by solvent extraction. In this study, an aqueous thermal pretreatment was explored as a method for lysing the cell wall of the oleaginous yeast Rhodotorula toruloides for lipid recovery. Hydrothermal pretreatment for 60 min at 121 °C with a dry cell weight of 7% (w/v) in the yeast slurry led to a recovery of 84.6 ± 3.2% (w/w) of the total lipids when extracted with organic solvents. The conventional sonication and acid-assisted thermal cell lysis led to a lipid recovery yield of 99.8 ± 0.03% (w/w) and 109.5 ± 1.9% (w/w), respectively. The fatty acid profiles of the hydrothermally pretreated cells and freeze-dried control were similar, suggesting that the thermal lysis of the cells did not degrade the lipids. This work demonstrates that hydrothermal pretreatment of yeast cell slurry at 121 °C for 60 min is a robust and sustainable method for cell conditioning to extract intracellular microbial lipids for biofuel production and provides a baseline for further scale-up and process integration.

Conversion↗
DOE OSTI2023

Integration of Yeast Episomal/Integrative Plasmid Causes Genotypic and Phenotypic Diversity and Improved Sesquiterpene Production in Metabolically Engineered Saccharomyces cerevisiae

The variability in phenotypic outcomes among biological replicates in engineered microbial factories presents a captivating mystery. Establishing the association between phenotypic variability and genetic drivers is important to solve this intricate puzzle. Here, we applied a previously developed auxin-inducible depletion of hexokinase 2 as a metabolic engineering strategy for improved nerolidol production in Saccharomyces cerevisiae, and biological replicates exhibit a dichotomy in nerolidol production of either 3.5 or 2.5 g L –1 nerolidol. Harnessing Oxford Nanopore’s long-read genomic sequencing, we reveal a potential genetic cause—the chromosome integration of a 2μ sequence-based yeast episomal plasmid, encoding the expression cassettes for nerolidol synthetic enzymes. This finding was reinforced through chromosome integration revalidation, engineering nerolidol and valencene production strains, and generating a diverse pool of yeast clones, each uniquely fingerprinted by gene copy numbers, plasmid integrations, other genomic rearrangements, protein expression levels, growth rate, and target product productivities. Τhe best clone in two strains produced 3.5 g L –1 nerolidol and ~0.96 g L –1 valencene. Comparable genotypic and phenotypic variations were also generated through the integration of a yeast integrative plasmid lacking 2μ sequences. Our work shows that multiple factors, including plasmid integration status, subchromosomal location, gene copy number, sesquiterpene synthase expression level, and genome rearrangement, together play a complicated determinant role on the productivities of sesquiterpene product. Integration of yeast episomal/integrative plasmids may be used as a versatile method for increasing the diversity and optimizing the efficiency of yeast cell factories, thereby uncovering metabolic control mechanisms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Machine learning reveals genes impacting oxidative stress resistance across yeasts

Reactive oxygen species (ROS) are highly reactive molecules encountered by yeasts during routine metabolism and during interactions with other organisms, including host infection. Here, we characterized the variation in resistance to ROS across the ancient yeast subphylum Saccharomycotina and used machine learning (ML) to identify gene families whose sizes were predictive of ROS resistance.

AI↗
DOE OSTI2024

Exploring Saccharomycotina Yeast Ecology Through an Ecological Ontology Framework

Yeasts in the subphylum Saccharomycotina are found across the globe in disparate ecosystems. A major aim of yeast research is to understand the diversity and evolution of ecological traits, such as carbon metabolic breadth, insect association, and cactophily. This includes studying aspects of ecological traits like genetic architecture or association with other phenotypic traits. Genomic resources in the Saccharomycotina have grown rapidly. Ecological data, however, are still limited for many species, especially those only known from species descriptions where usually only a limited number of strains are studied. Moreover, ecological information is recorded in natural language format limiting high throughput computational analysis. To address these limitations, we developed an ontological framework for the analysis of yeast ecology. A total of 1,088 yeast strains were added to the Ontology of Yeast Environments (OYE) and analyzed in a machine-learning framework to connect genotype to ecology. This framework is flexible and can be extended to additional isolates, species, or environmental sequencing data. Widespread adoption of OYE would greatly aid the study of macroecology in the Saccharomycotina subphylum.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Leveraging 13C-Labeling to Assign Molecular Formulas to Unknown Yeast Metabolites

Mass spectrometry analyses have identified tens of thousands of unknown small molecule-associated peaks in different biological specimens. Notably, even the simplest and best studied organisms like Escherichia coli and Saccharomyces cerevisiae yield thousands of unknown peaks. A key question is how many of these reflect actual novel endogenous metabolites. To explore this, Mahieu and Patti used complete 13 C -labeling in E. coli to credential peaks as biological. This reduced the number of unknowns by more than 90%. Here, we carry out similar uniform 13 C-labeling in the Baker’s yeast S. cerevisiae and two less-studied bioenergy-relevant yeasts Rhodotorula toruloides (lipid producer) and Issatchenkia orientalis (organic acid producer). Identification of unknown metabolite peaks and their molecular formulas is facilitated through software tailored for 13 C labeling data and resulting knowledge of carbon atom count. A classification model evaluates the plausibility of each candidate formula, with peaks lacking plausible candidate formulas unlikely to reflect metabolite molecular ions. This approach prioritizes about one hundred candidate abundant unknown metabolites with logical molecular formulas. Most of these are species-specific rather than conserved across yeasts, and more are found in the nonmodel yeasts than S. cerevisiae. Thus, 13 C-labeling data on unknown metabolites highlights the potential for discovering new metabolites and pathways in nonmodel yeasts.

Carbon↗
DOE OSTI2026

The oleaginous yeast Cutaneotrichosporon oleaginosum modifies corn stover alkali lignin

The current paradigm in synthetic biology for lignin bioconversion platforms includes primarily bacteria and filamentous fungi. Yeast are notoriously understudied for their role in lignin degradation and utilization, despite their ubiquity in saprophytic microbial communities. A few publications report lignin-modifying yeasts, but investigations to date have relied on model aromatic compounds or lignin-containing substrates replete with other carbon sources. In this work, we use a suite of analytical tools to evaluate interactions between corn stover-extracted lignin and the oleaginous yeast Cutaneotrichosporon oleaginosum. Notably, 2D-NMR analysis showed a significant decrease in the H-lignin component as well as resinol (β-β) and phenylcoumaran (β-5) linkages. Using super-resolution fluorescence microscopy, we demonstrated that this yeast may uptake polymeric lignin and/or undertakes interactions at the cellular envelope. To explore mechanisms of lignin modification, transport, and aromatics catabolism, extensive secretomics and proteomics analyses were conducted. Compared to carbon-limited glucose and “No Carbon” controls, several putative laccases, quinone reductases, superoxide dismutases, and glyoxal/oxalate oxidases were upregulated in the lignin condition. Excitingly, two ferric reductases and an oxalate exchanger were only observed in the lignin condition. These results indicate that C. oleaginosum may perform extracellular quinone redox cycling to generate lignin-modifying reactive oxygen species. These findings enhance our understanding of yeast-lignin interactions and provide valuable insights for validation studies and metabolic engineering.

09 BIOMASS FUELS↗
DOE OSTI2023

The cell morphological diversity of Saccharomycotina yeasts

The ~1 200 known species in subphylum Saccharomycotina are a highly diverse clade of unicellular fungi. During its lifecycle, a typical yeast exhibits multiple cell types with various morphologies; these morphologies vary across Saccharomycotina species. Here, we synthesize the evolutionary dimensions of variation in cellular morphology of yeasts across the subphylum, focusing on variation in cell shape, cell size, type of budding, and filament production. Examination of 332 representative species across the subphylum revealed that the most common budding cell shapes are ovoid, spherical, and ellipsoidal, and that their average length and width is 5.6 µm and 3.6 µm, respectively. 58.4% of yeast species examined can produce filamentous cells, and 87.3% of species reproduce asexually by multilateral budding, which does not require utilization of cell polarity for mitosis. Interestingly, ~1.8% of species examined have not been observed to produce budding cells, but rather only produce filaments of septate hyphae and/or pseudohyphae. 76.9% of yeast species examined have sexual cycle descriptions, with most producing one to four ascospores that are most commonly hat-shaped (37.4%). Systematic description of yeast cellular morphological diversity and reconstruction of its evolution promises to enrich our understanding of the evolutionary cell biology of this major fungal lineage.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Switchgrass Steroidal Saponins Reduce Fungal Disease but Decrease Yeast Fermentation Yield

Increasing the production of bioproducts from lignocellulosic feedstocks requires improvement in both field production and biorefinery efficiency. When plant traits arise that improve field production but decrease biofuel yield, these trade-offs can represent challenges in the entire production process. To examine trade-offs between field and production traits, we examined factors underlying switchgrass resistance to fungal rust pathogens in field conditions and factors that impede yeast fermentation in the lab using repeated measurements on a switchgrass genetic diversity panel. We found that the same switchgrass genotypes that showed high fungal pathogen resistance also showed recalcitrance to yeast fermentation. These switchgrass genotypes were mostly from the Atlantic genetic group, which had high levels of specialized metabolites of the saponin class. Among 1589 metabolites identified through metabolomics, we found that saponins were among the most likely to explain variation in both rust infection and fermentation yield using random forest feature selection, and that only four of these were sufficient to explain 57.9% of the variation in rust susceptibility. Through follow-up testing in recalcitrant biomass, we found that the bacterium Zymomonas mobilis does not suffer the same inhibition as the yeast Saccharomyces cerevisiae, and that the addition of ergosterol (thought to be the fungal cellular target of saponin inhibition) rescues yeast fermentation. Several lines of evidence point to a central role for saponins as key metabolites protecting switchgrass from fungal pathogens and interfering with yeast fermentation, underscoring an ongoing need for collaboration between plant breeders and biofuel production scientists.

VanWallendael, Acer [North Carolina State Universi↗
DOE OSTI2025

Unique trajectory of gene family evolution from genomic analysis of nearly all known species in an ancient yeast lineage

Gene gains and losses are a major driver of genome evolution; their precise characterization can provide insights into the origin and diversification of major lineages. Here, we examined gene family evolution of 1154 genomes from nearly all known species in the medically and technologically important yeast subphylum Saccharomycotina. We found that yeast gene family evolution differs from that of plants, animals, and filamentous ascomycetes, and is characterized by smaller overall gene numbers yet larger gene family sizes for a given gene number. Faster-evolving lineages (FELs) in yeasts experienced significantly higher rates of gene losses—commensurate with a narrowing of metabolic niche breadth—but higher speciation rates than their slower-evolving sister lineages (SELs). Gene families most often lost are those involved in mRNA splicing, carbohydrate metabolism, and cell division and are likely associated with intron loss, metabolic breadth, and non-canonical cell cycle processes. Our results highlight the significant role of gene family contractions in the evolution of yeast metabolism, genome function, and speciation, and suggest that gene family evolutionary trajectories have differed markedly across major eukaryotic lineages.

Comparative Genomics↗