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All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Rhodotorula toruloides Nitrogen Limitation PTM Profiling Multi-Omics (TZ-DP1)

The purpose of this experiment was to evaluate the regulatory stress response of Oleaginous yeast species Rhodotorula toruloides NBRC 0880 (JGI strain IFO0880 v4.0) under nitrogen-rich and nitrogen-limited conditions over time. Time course experimental samples (0, 24, 48, and 72 hours after inoculation) were prepared using a semi-automated multi-PTM proteomic approach, using tandem mass tag 18-plex (TMT18), and lipidome remodeling for downstream multi-omics analysis. Processed datasets are openly accessible from PNNL DataHub and contain secondary processed proteomic (redox, phospho, and global TMT) and lipidomic (positive and negative ion mode) results files and experimental design metadata.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Rhodotorula toruloides Nitrogen Limitation PTM Profiling Multi-Omics (TZ-DP1).

The purpose of this experiment was to evaluate the regulatory stress response of Oleaginous yeast species Rhodotorula toruloides NBRC 0880 (JGI strain IFFO0880 v4.0) under nitrogen-rich and nitrogen-limited conditions over time. Time course experimental samples (24, 48, and 72 hours after inoculation) were prepared using a semi-automated multi-PTM proteomic approach, using tandem mass tag 18-plex (TMT18), and lipidome remodeling for downstream multi-omics analysis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Data for Metabolic Engineering of Oleaginous Yeast Rhodotorula toruloides for Overproduction of Triacetic Acid Lactone

The plant-sourced polyketide triacetic acid lactone (TAL) has been recognized as a promising platform chemical for the biorefinery industry. However, its practical application was rather limited due to low natural abundance and inefficient cell factories for biosynthesis. Here, we report the metabolic engineering of oleaginous yeast Rhodotorula toruloides for TAL overproduction. We first introduced a 2-pyrone synthase gene from Gerbera hybrida (GhPS) into R. toruloides and investigated the effects of different carbon sources on TAL production. We then systematically employed a variety of metabolic engineering strategies to increase the flux of acetyl-CoA by enhancing its biosynthetic pathways and disrupting its competing pathways. We found that overexpression of ATP-citrate lyase (ACL1) improved TAL production by 45% compared to the GhPS overexpressing strain, and additional overexpression of acetyl-CoA carboxylase (ACC1) further increased TAL production by 29%. Finally, we characterized the resulting strain I12-ACL1-ACC1 using fed-batch bioreactor fermentation in glucose or oilcane juice medium with acetate supplementation and achieved a titer of 28 or 23 g/L TAL, respectively. This study demonstrates that R. toruloides is a promising host for the production of TAL and other acetyl-CoA-derived polyketides from low-cost carbon sources.

Conversion↗
DOE OSTI2025

Nitrogen limitation causes a seismic shift in redox state and phosphorylation of proteins implicated in carbon flux and lipidome remodeling in Rhodotorula toruloides

Background: Oleaginous yeast are prodigious producers of oleochemicals, offering alternative and secure sources for applications in foodstuff, skincare, biofuels, and bioplastics. Nitrogen starvation is the primary strategy used to induce oil accumulation in oleaginous yeast as part of a global stress response. While research has demonstrated that post-translational modifications (PTMs), including phosphorylation and protein cysteine thiol oxidation (redox PTMs), are involved in signaling pathways that regulate stress responses in metazoa and algae, their role in oleaginous yeast remain understudied and unexplored. Results: Towards linking the yeast oleaginous phenotype to protein function, we integrated lipidomics, redox proteomics, and phosphoproteomics to investigate Rhodotorula toruloides under nitrogen-rich and starved conditions over time. Our lipidomics results unearthed interactions involving sphingolipids and cardiolipins with ER stress and mitophagy. Our redox and phosphoproteomics data highlighted the roles of the AMPK, TOR, and calcium signaling pathways in regulation of lipogenesis, autophagy, and oxidative stress response. As a first, we also demonstrated that lipogenic enzymes including fatty acid synthase are modified as a consequence of shifts in cellular redox states due to nutrient availability. Conclusions: We conclude that lipid accumulation is largely a consequence of carbon rerouting and autophagy governed by changes to PTMs, and not increases in the abundance of enzymes involved in central carbon metabolism and fatty acid biosynthesis. Our systems-level approach sets the stage for acquiring multidimensional data sets for protein structural modeling and predicting the functional relevance of PTMs using Artificial Intelligence/Machine Learning (AI/ML). Coupled to those bioinformatics approaches, the putative PTM switches that we delineate will enable advanced metabolic engineering strategies to decouple lipid accumulation from nitrogen limitation.

Lipid Signalling↗
DOE OSTI2026

Next-Generation Sequencing Data from a CUT&RUN Study of R. toruloides IFO0880 Cse4 and Orc1 Binding Sites

Rhodotorula toruloides has been increasingly explored as a host for bioproduction of lipids, fatty acid derivatives and terpenoids. Various genetic tools have been developed, but neither a centromere nor an autonomously replicating sequence (ARS), both necessary elements for stable episomal plasmid maintenance, has yet been reported. In this study, cleavage under targets and release using nuclease (CUT&RUN), a method used for genome-wide mapping of DNA–protein interactions, was used to identify R. toruloides IFO0880 genomic regions associated with the centromeric histone H3 protein Cse4, a marker of centromeric DNA. Fifteen putative centromeres ranging from 8 to 19 kb in length were identified and analyzed, and four were tested for, but did not show, ARS activity. These centromeric sequences contained below average GC content, corresponded to transcriptional cold spots, were primarily nonrepetitive and shared some vestigial transposon-related sequences but otherwise did not show significant sequence conservation. Future efforts to identify an ARS in this yeast can utilize these centromeric DNA sequences to improve the stability of episomal plasmids derived from putative ARS elements.

Genome Engineering↗
DOE OSTI2025

Zhao-Group/Integration-Sites-IFO0880

Integration sites for Rhodotorula toruloides strain NBRC 0880. This repository accompanies the work "Discovery, characterization, and application of chromosomal integration sites for stable heterologous gene expression in Rhodotorula toruloides". Add scripts and ifo0880 folders to code and data folder of "CRISPR-COPIES" repository.

Boob, Aashutosh↗
DOE OSTI2023

Engineering transcriptional regulation of pentose metabolism in Rhodosporidium toruloides for improved conversion of xylose to bioproducts

Abstract Efficient conversion of pentose sugars remains a significant barrier to the replacement of petroleum-derived chemicals with plant biomass-derived bioproducts. While the oleaginous yeast Rhodosporidium toruloides (also known as Rhodotorula toruloides ) has a relatively robust native metabolism of pentose sugars compared to other wild yeasts, faster assimilation of those sugars will be required for industrial utilization of pentoses. To increase the rate of pentose assimilation in R. toruloides, we leveraged previously reported high-throughput fitness data to identify potential regulators of pentose catabolism. Two genes were selected for further investigation, a putative transcription factor (RTO4_12978, Pnt1) and a homolog of a glucose transceptor involved in carbon catabolite repression (RTO4_11990). Overexpression of Pnt1 increased the specific growth rate approximately twofold early in cultures on xylose and increased the maximum specific growth by 18% while decreasing accumulation of arabitol and xylitol in fast-growing cultures. Improved growth dynamics on xylose translated to a 120% increase in the overall rate of xylose conversion to fatty alcohols in batch culture. Proteomic analysis confirmed that Pnt1 is a major regulator of pentose catabolism in R. toruloides. Deletion of RTO4_11990 increased the growth rate on xylose, but did not relieve carbon catabolite repression in the presence of glucose. Carbon catabolite repression signaling networks remain poorly characterized in R. toruloides and likely comprise a different set of proteins than those mainly characterized in ascomycete fungi.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Acetate-based biological platforms: Bridging carbon dioxide utilization and high-value bioproduct production in oleaginous yeasts

Acetate is emerging as a promising two-carbon substrate in the circular bioeconomy, bridging the gap between single-carbon sources and high-value biofuels and bioproducts. This review examines the key pathways for acetate production, including the electrochemical reduction of carbon dioxide, syngas fermentation, and biological acetogenesis. It focuses on acetate metabolism in oleaginous yeasts, such as Yarrowia lipolytica and Rhodotorula toruloides, which efficiently convert acetate-derived acetyl-CoA units into diverse bioproducts such as lipids, fatty alcohols, triacetic acid lactone, and carotenoids. Recent advances in metabolic engineering, transcriptomics, and metabolic flux analysis have improved the understanding of acetate assimilation in these organisms, thereby increasing their potential for industrial applications. In addition, the feasibility of a biological gas-to-liquid platform that utilizes acetate as a central intermediate for scalable biomanufacturing is discussed. Integrating acetate utilization with sustainable production strategies offers a promising path to advance the bio-based economy. Using acetate as a versatile metabolic intermediate enables the conversion of industrial emissions into biofuels and bioproducts while avoiding the energetic and toxicity constraints associated with direct fermentation of gaseous substrates.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Green pretreatment strategies for enhanced microbial lipid fermentation and synergistic high-quality lignin recovery for next-generation integrated biorefineries

Miscanthus × giganteus (Mxg) is a warm-season perennial grass being commercialized as a biomass feedstock for temperate farms. Three process strategies were compared for converting Mxg into single-cell oil and extracted lignin intermediates, as hydrothermal (HT) processing and two natural deep eutectic solvents (NADES), ChCl:LA (choline chloride:lactic acid) and ChCl:Gly (choline chloride:glycerol). Pretreatments were performed at 10% and 50% solids loading at 140 °C, 2 h; HT: 190 °C, 10 min. Enzymatic hydrolysates, generated at 10% solids using washed and unwashed biomass, were evaluated for microbial lipid production. Maximum glucose conversions from washed biomass reached 83.5% (ChCl:LA), 52.7% (ChCl:Gly), and 74.0% (HT). Remarkably, NADES-derived hydrolysates effectively replaced refined sugars for the cultivation of the oleaginous yeast Rhodotorula toruloides, achieving ∼51% higher biomass (OD 90.7) and lipid titers of 19.36 g/L within 45 h using a two-stage fermentation strategy. Lipid contents ranged from 34.5–44.5% dry weight, demonstrating reduced reliance on purified sugars. Beyond carbohydrate valorization, ChCl:LA pretreatment enabled high-purity lignin recovery (>89%) in a lignin-first strategy. Structural analyses (2D-HSQC and ³¹P NMR) showed syringyl (78.15%), guaiacyl (15.15%), and p-hydroxyphenyl (6.41%) units, with higher phenolic hydroxyl content (0.91 mmol/g) and a lower S/G ratio (0.19) than HT lignin (0.87 mmol/g, S/G 0.22). These attributes favor downstream lignin depolymerization into low-molecular-weight aromatics. Overall, NADES pretreatment simultaneously enhances microbial lipid yields and recovers high-quality lignin, advancing the economic feasibility of renewable diesel and sustainable aviation fuel (SAF) production from bioenergy crops within an integrated biorefinery framework.

2D HSQC↗
DOE OSTI2026

Lipid accumulation in nitrogen and phosphorus-limited yeast is caused by less growth-related dilution

Oleaginous yeasts are used commercially to produce oleochemicals and hold potential also for biodiesel production. In response to nitrogen or phosphorous limitation, oleaginous yeasts accumulate lipids in the form of triacylglycerols. Previous work has investigated potential mechanisms by which nutrient limitation induces lipid biosynthesis without verifying whether lipid biosynthesis flux is actually enhanced. Here, in this work, we show, using 13C-glucose tracing, that in nitrogen or phosphorous limitation, lipid accumulation occurs without consistent increases in biosynthetic flux. Instead, the main driver of increased lipid pools is decreased growth-related dilution. This conclusion holds across two divergent oleaginous yeasts: Rhodotorula toruloides and Yarrowia lipolytica . Quantitative proteomics shows a substantial proteome reallocation in response to nitrogen and phosphorous limitation, with ribosomal proteins strongly downregulated, while lipid enzymes are preserved but not consistently upregulated in absolute quantity. Thus, nutrient limitation, rather than triggering greatly enhanced lipid synthesis, results in roughly sustained lipid enzyme levels and biosynthetic flux. Due to slower lipid dilution by cell division, this suffices to drive marked lipid accumulation.

09 BIOMASS FUELS↗
DOE OSTI2025

Production of a δ-Lactam from Glucose through Integrating Biological and Chemical Catalysis

We present a new strategy for the production of a δ-lactam from glucose that integrates biological production of triacetic acid lactone (TAL, 4-hydroxy-6-methyl-2H-2-one) with catalytic transformation of TAL into 6-methylpiperidin-2-one (MPO) through metabolic engineering, isomerization, amination, and catalytic hydrogenation/hydrogenolysis. We developed a sustainable and antibiotic-free fed-batch fermentation using genetically modified Rhodotorula toruloides IFO0880. This process achieved a yield of 2-hydroxy-6-methyl-4H-pyran-4-one (2H4P) at 0.05 g/g of glucose, corresponding to a 9.9 g/L titer. By adjusting the pH of the fermentation broth to 2, 2H4P was quantitatively converted into TAL. The TAL in the fermentation broth was directly converted by aminolysis into 4-hydroxy-6-methylpyridin-2(1H)-one (HMPO), which achieved an 18.5% yield with 94.3% purity. The HMPO yield was lower in the fermentation broth than in a clean feedstock (32.2%), suggesting that the biological impurities are inhibitors in this reaction. Further investigation revealed that lower pH levels and reduced TAL concentrations in the fermentation broth significantly decreased HMPO yields. Subsequently, the precipitated HMPO was filtered and dried and then subjected to the final catalytic conversion in H2O solvent, achieving a MPO yield of 91.8%. Furthermore, this integrated approach demonstrated the direct use of TAL in the filtered aqueous fermentation broth without the need to isolate TAL.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
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 OSTI2024

Mixed and membrane-separated culturing of synthetic cyanobacteria-yeast consortia reveals metabolic cross-talk mimicking natural cyanolichens

Metabolite exchange mediates crucial interactions in microbial communities, significantly impacting global carbon and nitrogen cycling. Understanding these chemically-mediated interactions is essential for elucidating natural community functions and developing engineered synthetic communities. This study investigated membrane-separated bioreactors (mBRs) as a novel tool to identify transient metabolites and their producers/consumers in mixed microbial communities. We compared three co-culture methods (direct mixed, 2-chamber mBR, and 3-chamber mBR) to grow a synthetic binary community of the cyanobacterium Synechococcus elongatus PCC 7942 and the fungus Rhodotorula toruloides NBRC 0880, as well as axenic S. elongatus. Despite not being natural lichen constituents, these organisms exhibited interactions resembling those in cyanolichens. S. elongatus fixed CO 2 into sugars as the primary shared metabolite, while R. toruloides secreted various biochemicals, predominantly sugar alcohols, mirroring the metabolite exchange observed in natural lichens. The mBR systems successfully captured metabolite gradients and revealed rapidly consumed compounds, including TCA cycle intermediates and amino acids. Our approach demonstrated that the 2-chamber mBR optimally balanced metabolite exchange and growth dynamics. This study provides insights into cross-species metabolic interactions and presents a valuable tool for investigating and engineering synthetic microbial communities with potential applications in biotechnology and environmental science.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

YEASTRACT+: a portal for the exploitation of global transcription regulation and metabolic model data in yeast biotechnology and pathogenesis

Abstract YEASTRACT+ (http://yeastract-plus.org/) is a tool for the analysis, prediction and modelling of transcription regulatory data at the gene and genomic levels in yeasts. It incorporates three integrated databases: YEASTRACT (http://yeastract-plus.org/yeastract/), PathoYeastract (http://yeastract-plus.org/pathoyeastract/) and NCYeastract (http://yeastract-plus.org/ncyeastract/), focused on Saccharomyces cerevisiae, pathogenic yeasts of the Candida genus, and non-conventional yeasts of biotechnological relevance. In this release, YEASTRACT+ offers upgraded information on transcription regulation for the ten previously incorporated yeast species, while extending the database to another pathogenic yeast, Candida auris. Since the last release of YEASTRACT+ (January 2020), a fourth database has been integrated. CommunityYeastract (http://yeastract-plus.org/community/) offers a platform for the creation, use, and future update of YEASTRACT-like databases for any yeast of the users’ choice. CommunityYeastract currently provides information for two Saccharomyces boulardii strains, Rhodotorula toruloides NP11 oleaginous yeast, and Schizosaccharomyces pombe 972h-. In addition, YEASTRACT+ portal currently gathers 304 547 documented regulatory associations between transcription factors (TF) and target genes and 480 DNA binding sites, considering 2771 TFs from 11 yeast species. A new set of tools, currently implemented for S. cerevisiae and C. albicans, is further offered, combining regulatory information with genome-scale metabolic models to provide predictions on the most promising transcription factors to be exploited in cell factory optimisation or to be used as novel drug targets. The expansion of these new tools to the remaining YEASTRACT+ species is ongoing.

Teixeira, Miguel Cacho (ORCID:0000000256766174)↗
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↗