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All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

Data for Development, Optimization, and Application of an Episomal Plasmid System for Rhodotorula toruloides

Rhodotorula toruloides is an emerging oleaginous yeast with strong potential as a microbial cell factory for the production of acetyl-CoA-derived bioproducts. However, engineering of this organism has been limited by the absence of a functional episomal plasmid system, a foundational genetic tool for rapid gene expression, pathway testing, and CRISPR-based genome engineering. Here, we report the first episomal plasmid system for R. toruloides . Through systematic screening of candidate autonomously replicating sequences (ARSs) from diverse sources, we identified multiple functional ARS elements and selected C63F4, a fragment derived from Contig 63 of R. toruloides CBS14, because of its stable performance. The resulting pC63F4 plasmid was maintained episomally, supported GFP reporter expression, exhibited a copy number of 2.39 ± 0.13, and showed good stability during long term cultivation. To overcome poor transformation efficiency, we developed a Cre-loxP-mediated in vivo re-circularization strategy that enabled reliable delivery of the episomal plasmid. Using this improved system, we demonstrated functional episomal expression of metabolic engineering genes and multi-gene pathways for the production of triacetic acid lactone, fatty alcohols, and limonene. Finally, we leveraged this platform to establish a redesigned CRISPR system that enables seamless genome editing in R. toruloides for the first time, while also simplifying marker recycling. Together, this work establishes a long-needed episomal plasmid platform and associated CRISPR toolkit that will accelerate metabolic engineering, synthetic biology, and fundamental studies in R. toruloides .

Gene Editing↗
DOE OSTI2023

Data for Determining Mating Type and Ploidy in Rhodotorula toruloides and its Effect on Growth on Sugars from Lignocellulosic Biomass

Rhodotorula toruloides is being developed for the use in industrial biotechnology processes because of its favorable physiology. This includes its ability to produce and store large amounts of lipids in the form of intracellular lipid bodies. Nineteen strains were characterized for mating type, ploidy, robustness for growth, and accumulation of lipids on inhibitory switchgrass hydrolysate (SGH). Mating type was determined using a novel polymerase chain reaction (PCR)-based assay, which was validated using the classical microscopic test. Three of the strains were heterozygous for mating type (A1/A2). Ploidy analysis revealed a complex pattern. Two strains were triploid, eight haploid, and eight either diploid or aneuploid. Two of the A1/A2 strains were compared to their parents for growth on 75%v/v concentrated SGH. The A1/A2 strains were much more robust than the parental strains, which either did not grow or had extended lag times. The entire set was evaluated in 60%v/v SGH batch cultures for growth kinetics and biomass and lipid production. Lipid titers were 2.33–9.40 g/L with a median of 6.12 g/L, excluding the two strains that did not grow. Lipid yields were 0.032–0.131 (g/g) and lipid contents were 13.5–53.7% (g/g). Four strains had significantly higher lipid yields and contents. One of these strains, which had among the highest lipid yield in this study (0.131 ± 0.007 g/g), has not been previously described in the literature.

Conversion↗
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 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 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 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 OSTI2021

Biosurfactant production by halophilic yeasts isolated from extreme environments in Botswana

ABSTRACT Nine morphologically distinct halophilic yeasts were isolated from Makgadikgadi and Sua pans, as pristine and extreme environments in Botswana. Screening for biosurfactant production showed that Rhodotorula mucilaginosa SP6 and Debaryomyces hansenii MK9 exhibited the highest biosurfactant activity using Xanthocercis zambesiaca seed powder as a novel and alternative inexpensive carbon substrate. Chemical characterization of the purified biosurfactants by Fourier Transform Infra-Red spectroscopy suggested that the biosurfactant from R. mucilaginosa SP6 was a rhamnolipid-type whereas the biosurfactant from D. hansenii MK9 was a sophorolipid-type. The two biosurfactants exhibited antimicrobial activities against eight pathogenic bacteria and fungal strains (Proteus vulgaris, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Micrococcus luteus, Cryptococcus neoformans, Candida albicans and Aspergilus niger). The sophorolopid-type biosurfactant was found to be the most potent among the antimicrobial drug resistant strains tested. The findings open up prospects for the development of environmentally friendly antimicrobial drugs that use an inexpensive source of carbon to reduce the costs associated with the production of biosurfactants.

Loeto, Daniel↗
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 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↗