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At least 163 records · Page 9
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Metabolic engineering of oleaginous yeast Rhodotorula toruloides for overproduction of triacetic acid lactone

Abstract 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.

2‐pyrone synthase↗
DOE OSTI2025

Taxogenomic Analysis of a Novel Yeast Species, Lachancea rosae Sp. Nov. F.A., Isolated From the Wild Rose Rosa californica

A novel Saccharomycotina yeast strain, yHQL494, was isolated from the rose hip of the wild rose Rosa californica from Castle Crags State Park, California, USA. Phylogenetic analyses of both whole genome data and the sequences from the D1/D2 region of the large ribosomal subunit (LSU) rRNA gene placed strain yHQL494 within the genus Lachancea and grouped it into a clade with Lachancea lanzarotensis and Lachancea meyersii. Taxogenomic analyses were conducted on publicly available genome sequences to gain a deeper insight into the carbon and nitrogen gene-trait associations across the Lachancea clade. The results of these analyses were found to be consistent across Lachancea species. Growth assays and microscopic analyses were conducted to determine the physiological characteristics of strain yHQL494, including the presence of hyphae or pseudohyphae, ascospore formation, fermentation abilities, and assimilation of carbon and nitrogen compounds. Based on the phenotypic and genomic characteristics of the strain yHQL494 T (=NRRL Y-64858 T , =CBS 18,574 T ), we propose a new species, Lachancea rosae sp. nov. f.a.

Bajgain, Yakendra [Villanova Univ., PA (United Sta↗
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 OSTI2022

Fission yeast Duf89 and Duf8901 are cobalt/nickel-dependent phosphatase–pyrophosphatases that act via a covalent aspartyl–phosphate intermediate

Domain of Unknown Function 89 (DUF89) proteins are metal-dependent phosphohydrolases. Exemplary DUF89 enzymes differ in their metal and phosphosubstrate preferences. Here, we interrogated the activities and structures of two DUF89 paralogs from fission yeast—Duf89 and Duf8901. We find that Duf89 and Duf8901 are cobalt/nickel-dependent phosphohydrolases adept at hydrolyzing p-nitrophenylphosphate and PP i . Crystal structures of metal-free Duf89 and Co 2+ -bound Duf8901 disclosed two enzyme conformations that differed with respect to the position of a three-helix module, which is either oriented away from the active site in Duf89 or forms a lid over the active site in Duf8901. Lid closure results in a 16 Å movement of Duf8901 Asp195, vis-à-vis Asp199 in Duf89, that brings Asp195 into contact with an octahedrally coordinated cobalt. Reaction of Duf8901 with BeCl 2 and NaF in the presence of divalent cations Co 2+ , Ni 2+ , or Zn 2+ generated covalent Duf8901-(Asp248)–beryllium trifluoride (BeF 3 )•Co 2+ , Duf8901-(Asp248)–BeF 3 •Ni 2+ , or Duf8901-(Asp248)–BeF 3 •Zn 2+ adducts, the structures of which suggest a two-step catalytic mechanism via formation and hydrolysis of an enzyme-(aspartyl)–phosphate intermediate. Alanine mutations of Duf8901 Asp248, Asn249, Lys401, Asp286, and Asp195 that interact with BeF 3 •Co 2+ squelched p-nitrophenylphosphatase activity. A 1.8 Å structure of a Duf8901-(Asp248)–AlF 4 –OH 2 •Co 2+ transition-state mimetic suggests an associative mechanism in which Asp195 and Asp363 orient and activate the water nucleophile. Whereas deletion of the duf89 gene elicited a phenotype in which expression of phosphate homeostasis gene pho1 was derepressed, deleting duf8901 did not, thereby hinting that the DUF89 paralogs have distinct functional repertoires in vivo.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Metabolic engineering of low-pH-tolerant non-model yeast, Issatchenkia orientalis , for production of citramalate

Methyl methacrylate (MMA) is an important petrochemical with many applications. However, its manufacture has a large environmental footprint. Combined biological and chemical synthesis (semisynthesis) may be a promising alternative to reduce both cost and environmental impact, but strains that can produce the MMA precursor (citramalate) at low pH are required. A non-conventional yeast, Issatchenkia orientalis, may prove ideal, as it can survive extremely low pH. Here, we demonstrate the engineering of I. orientalis for citramalate pro- duction. Using sequence similarity network analysis and subsequent DNA synthesis, we selected a more active citramalate synthase gene (cimA) variant for expression in I. orientalis. We then adapted a piggyBac transposon system for I. orientalis that allowed us to simultaneously explore the effects of different cimA gene copy numbers and integration locations. A batch fermentation showed the genome-integrated-cimA strains produced 2.0 g/L citramalate in 48 h and a yield of up to 7% mol citramalate/mol consumed glucose. These results demonstrate the potential of I. orientalis as a chassis for citramalate production.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

The oleaginous yeast Rhodosporidium toruloides engineered for biomass hydrolysate-derived (E)-α-bisabolene production

The oleaginous yeast Rhodosporidium toruloides has been exploited for many bioproducts, including several terpenes, owing to its oleaginous nature and biomass inhibitor tolerance. Here, we built upon previous (E)-a-bisabolene work by iteratively stacking the complete mevalonate pathway from Saccharomyces cerevisiae onto a multicopy bisabolene synthase parent strain. Metabolomics and proteomics verified heterologous pathway expression and identified metabolic bottlenecks at three intermediate steps, with candidate feedback-resistant mevalonate kinases screening improving titers 15%. Subtle differences in codon optimization, and preliminary attenuation of competing flux toward lipids resulted in 6-fold, 7-fold higher titers relative to controls, respectively. Media optimization led to modest improvements, with zinc identified as the most promising at 10% titer improvement. Ultimately, high-performance strains were cultivated with corn-stover biomass hydrolysate in microtiter plates at 300g/L total sugar, achieving 20.8g/L bisabolene, the highest reported titer in the literature. A 2L glucose minimal medium bioreactor achieved 19.3 g/L bisabolene and a literature-high productivity of 0.11 g/L/h.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Structure-Guided Engineering of a Scoulerine 9-O-Methyltransferase Enables the Biosynthesis of Tetrahydropalmatrubine and Tetrahydropalmatine in Yeast

Benzylisoquinoline alkaloids (BIAs) are an important class of plant natural products with diverse pharmacological properties. Microbial platforms can produce BIAs through heterologous biosynthesis more efficiently than native plant hosts and allow for the generation of currently inaccessible BIA biosynthetic intermediates and unnatural derivatives. However, much remains unknown regarding the structures, substrate scopes, and mechanisms of many of the enzymes involved in BIA biosynthesis, which hampers efforts toward engineering these enzymes to produce alternative products or act in non-native biosynthetic contexts. In this paper, we present multiple crystal structures of two scoulerine 9- O -methyltransferase (S9OMT) variants from Thalictrum flavum which catalyze an essential step in the biosynthesis of berberine. The crystal structures revealed the structural basis for substrate and cofactor recognition by Tf S9OMT and provide further insight into the structure and function of S9OMTs. A structural comparison of the Tf S9OMT and T. flavum norcoclaurine 6OMT ( Tf 6OMT) crystal structures identified important residues for enzyme regiospecificity, which were confirmed via mutagenesis and in vitro assays. Several Tf S9OMT mutants with expanded substrate scopes toward various 1-benzylisoquinoline substrates were generated. The rationally engineered Tf S9OMT mutants with altered regiospecificity were tested in a yeast-based scoulerine production platform and enabled de novo production of tetrahydropalmatrubine and tetrahydropalmatine.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Anti-Pdc1p Nanobody as a Genetically Encoded Inhibitor of Ethanol Production Enables Dual Transcriptional and Post-translational Controls of Yeast Fermentations

Microbial fermentation provides a sustainable method of producing valuable chemicals. Adding dynamic control to fermentations can significantly improve titers, but most systems rely on transcriptional controls of metabolic enzymes, leaving existing intracellular enzymes unregulated. This limits the ability of transcriptional controls to switch off metabolic pathways, especially when metabolic enzymes have long half-lives. We developed a two-layer transcriptional/post-translational control system for yeast fermentations. Specifically, the system uses blue light to transcriptionally activate the major pyruvate decarboxylase PDC1, required for cell growth and concomitant ethanol production. Switching to darkness transcriptionally inactivates PDC1 and instead activates the anti-Pdc1p nanobody, NbJRI, to act as a genetically encoded inhibitor of Pdc1p accumulated during the growth phase. This dual transcriptional/post-translational control improves the production of 2,3-BDO and citramalate by up to 100 and 92% compared to using transcriptional controls alone in dynamic two-phase fermentations. Furthermore, this study establishes the NbJRI nanobody as an effective genetically encoded inhibitor of Pdc1p that can enhance the production of pyruvate-derived chemicals.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A yeast platform for high-level synthesis of tetrahydroisoquinoline alkaloids

The tetrahydroisoquinoline (THIQ) moiety is a privileged substructure of many bioactive natural products and semi-synthetic analogs. Plants manufacture more than 3,000 THIQ alkaloids, including the opioids morphine and codeine. While microbial species have been engineered to synthesize a few compounds from the benzylisoquinoline alkaloid (BIA) family of THIQs, low product titers impede industrial viability and limit access to the full chemical space. Here we report a yeast THIQ platform by increasing production of the central BIA intermediate ( S )-reticuline to 4.6 g L –1 , a 57,000-fold improvement over our first-generation strain. We show that gains in BIA output coincide with the formation of several substituted THIQs derived from amino acid catabolism. We use these insights to repurpose the Ehrlich pathway and synthesize an array of THIQ structures. This work provides a blueprint for building diverse alkaloid scaffolds and enables the targeted overproduction of thousands of THIQ products, including natural and semi-synthetic opioids.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

The interplay of additivity, dominance, and epistasis on fitness in a diploid yeast cross

In diploid species, genetic loci can show additive, dominance, and epistatic effects. To characterize the contributions of these different types of genetic effects to heritable traits, we use a double barcoding system to generate and phenotype a panel of ~200,000 diploid yeast strains that can be partitioned into hundreds of interrelated families. This experiment enables the detection of thousands of epistatic loci, many whose effects vary across families. Here, we show traits are largely specified by a small number of hub loci with major additive and dominance effects, and pervasive epistasis. Genetic background commonly influences both the additive and dominance effects of loci, with multiple modifiers typically involved. The most prominent dominance modifier in our data is the mating locus, which has no effect on its own. Our findings show that the interplay between additivity, dominance, and epistasis underlies a complex genotype-to-phenotype map in diploids.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Macroevolutionary diversity of traits and genomes in the model yeast genus Saccharomyces

Species is the fundamental unit to quantify biodiversity. In recent years, the model yeast Saccharomyces cerevisiae has seen an increased number of studies related to its geographical distribution, population structure, and phenotypic diversity. However, seven additional species from the same genus have been less thoroughly studied, which has limited our understanding of the macroevolutionary events leading to the diversification of this genus over the last 20 million years. Here, we show the geographies, hosts, substrates, and phylogenetic relationships for approximately 1,800 Saccharomyces strains, covering the complete genus with unprecedented breadth and depth. We generated and analyzed complete genome sequences of 163 strains and phenotyped 128 phylogenetically diverse strains. This dataset provides insights about genetic and phenotypic diversity within and between species and populations, quantifies reticulation and incomplete lineage sorting, and demonstrates how gene flow and selection have affected traits, such as galactose metabolism. These findings elevate the genus Saccharomyces as a model to understand biodiversity and evolution in microbial eukaryotes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Decompartmentalization of the yeast mitochondrial metabolism to improve chemical production in Issatchenkia orientalis

Microbial production of chemicals may suffer from inadequate cofactor provision, a challenge further exacerbated in yeasts due to compartmentalized cofactor metabolism. Here, we perform cofactor engineering through the decompartmentalization of mitochondrial metabolism to improve succinic acid (SA) production in Issatchenkia orientalis. We localize the reducing equivalents of mitochondrial NADH to the cytosol through cytosolic expression of its pyruvate dehydrogenase (PDH) complex and couple a reductive tricarboxylic acid pathway with a glyoxylate shunt, partially bypassing an NADH-dependent malate dehydrogenase to conserve NADH. Cytosolic SA production reaches a titer of 104 g/L and a yield of 0.85 g/g glucose, surpassing the yield of 0.66 g/g glucose constrained by cytosolic NADH availability. Additionally, expressing cytosolic PDH, we expand our I. orientalis platform to enhance acetyl-CoA-derived citramalic acid and triacetic acid lactone production by 1.22- and 4.35-fold, respectively. Our work establishes I. orientalis as a versatile platform to produce markedly reduced and acetyl-CoA-derived chemicals.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

High-throughput platform for yeast morphological profiling predicts the targets of bioactive compounds

Abstract Morphological profiling is an omics-based approach for predicting intracellular targets of chemical compounds in which the dose-dependent morphological changes induced by the compound are systematically compared to the morphological changes in gene-deleted cells. In this study, we developed a reliable high-throughput (HT) platform for yeast morphological profiling using drug-hypersensitive strains to minimize compound use, HT microscopy to speed up data generation and analysis, and a generalized linear model to predict targets with high reliability. We first conducted a proof-of-concept study using six compounds with known targets: bortezomib, hydroxyurea, methyl methanesulfonate, benomyl, tunicamycin, and echinocandin B. Then we applied our platform to predict the mechanism of action of a novel diferulate-derived compound, poacidiene. Morphological profiling of poacidiene implied that it affects the DNA damage response, which genetic analysis confirmed. Furthermore, we found that poacidiene inhibits the growth of phytopathogenic fungi, implying applications as an effective antifungal agent. Thus, our platform is a new whole-cell target prediction tool for drug discovery.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Genetic circuit design automation for yeast

Cells can be programmed to monitor and react to their environment using genetic circuits. Design automation software maps a desired circuit function to a DNA sequence, a process that requires units of gene regulation (gates) that are simple to connect and behave predictably. This poses a challenge for eukaryotes due to their complex mechanisms of transcription and translation. To this end, we have developed gates for yeast (Saccharomyces cerevisiae) that are connected using RNA polymerase flux as the signal carrier and are insulated from each other and host regulation. They are based on minimal constitutive promoters (~120 base pairs), for which rules are developed to insert operators for DNA-binding proteins. Using this approach, in this work we constructed nine NOT/NOR gates with nearly identical response functions and 400-fold dynamic range. In circuits, they are transcriptionally insulated from each other by placing ribozymes downstream of terminators to block nuclear export of messenger RNAs resulting from RNA polymerase readthrough. Based on these gates, Cello 2.0 was used to build circuits with up to 11 regulatory proteins. A simple dynamic model predicts the circuit response over days. Genetic circuit design automation for eukaryotes simplifies the construction of regulatory networks as part of cellular engineering projects, whether it be to stage processes during bioproduction, serve as environmental sentinels or guide living therapeutics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Ancient co-option of LTR retrotransposons as yeast centromeres

Centromeres ensure accurate chromosome segregation, yet their DNA evolves rapidly across eukaryotes leaving the origins of new centromere architectures unclear. The brewer’s yeast Saccharomyces cerevisiae exemplifies this long-standing puzzle. Its centromeres shifted ancestrally from large, repeat-rich, epigenetically specified forms to the compact, genetically defined ‘point’ centromeres. How this transition occurred has remained unresolved6. Here we identify evolutionarily related ‘proto-point’ centromeres that provide a resolution to the evolutionary origins of point centromeres. Proto-point centromeres contain a single centromeric nucleosome positioned over an AT-rich core, accompanied by relaxed organization and sequence variability of flanking cis-elements. In two species, these proto-point centromeres lie within retrotransposon-derived repeat clusters, linking ancestral repeat-rich centromeres to genetically encoded ones. Comparative and phylogenetic analyses indicate that proto-point and point centromeres evolved in an ancestor with retrotransposon-rich centromeres. These results identify long-terminal-repeat retrotransposons, specifically Ty5 sequences, as the genetic substrate for point-centromere evolution and provide a mechanistic route by which an epigenetic centromere can become genetically specified. More broadly, they show how selfish elements can be co-opted to perform essential chromosomal functions.

Haase, Max A. B. [Max Planck Institute of Molecula↗
DOE OSTI2022

Glucose feeds the tricarboxylic acid cycle via excreted ethanol in fermenting yeast

Ethanol and lactate are typical waste products of glucose fermentation. In mammals, glucose is catabolized by glycolysis into circulating lactate, which is broadly used throughout the body as a carbohydrate fuel. Individual cells can both uptake and excrete lactate, uncoupling glycolysis from glucose oxidation. Here we show that similar uncoupling occurs in budding yeast batch cultures of Saccharomyces cerevisiae and Issatchenkia orientalis. Even in fermenting S. cerevisiae that is net releasing ethanol, media 13 C-ethanol rapidly enters and is oxidized to acetaldehyde and acetyl-CoA. This is evident in exogenous ethanol being a major source of both cytosolic and mitochondrial acetyl units. 2 H-tracing reveals that ethanol is also a major source of both NADH and NADPH high-energy electrons, and this role is augmented under oxidative stress conditions. Furthermore, uncoupling of glycolysis from the oxidation of glucose-derived carbon via rapidly reversible reactions is a conserved feature of eukaryotic metabolism.

36 MATERIALS SCIENCE↗
DOE OSTI2023

Biosynthesis of natural and halogenated plant monoterpene indole alkaloids in yeast

Monoterpenoid indole alkaloids (MIAs) represent a large class of plant natural products with marketed pharmaceutical activities against a wide range of indications, including cancer, malaria and hypertension. Halogenated MIAs have shown improved pharmaceutical properties; however, synthesis of new-to-nature halogenated MIAs remains a challenge. Here we demonstrate a platform for de novo biosynthesis of two MIAs, serpentine and alstonine, in baker’s yeast Saccharomyces cerevisiae and deploy it to systematically explore the biocatalytic potential of refactored MIA pathways for the production of halogenated MIAs. From this, we demonstrate conversion of individual haloindole derivatives to a total of 19 different new-to-nature haloserpentine and haloalstonine analogs. Furthermore, by process optimization and heterologous expression of a modified halogenase in the microbial MIA platform, we document de novo halogenation and biosynthesis of chloroalstonine. Together, this study highlights a microbial platform for enzymatic exploration and production of complex natural and new-to-nature MIAs with therapeutic potential.

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↗