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At least 163 records · Page 9
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

A mass spectrometry-based high-throughput screening method for engineering fatty acid synthases with improved production of medium-chain fatty acids

Microbial cell factories have been extensively engineered to produce free fatty acids (FFAs), key components of crucial nutrients, soaps, industrial chemicals, and fuels. Yet, our ability to control the specificity of microbially synthesized FFAs is still limited for medium-chain fatty acids (MCFAs), which is mainly due to lack of high-throughput approaches for FFA analysis. Here we report a mass spectrometry (MS)-based method for rapid profiling of MCFAs in Saccharomyces cerevisiae by using membrane lipids as a proxy. Specially, matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) MS was used to detect shorter acyl chain phosphatidylcholines from membrane lipids and the m/z peak ratio at 730 and 758 was used as an index for improved MCFA production. This yeast colony-based method can be performed at a rate of ~2 second per sample, which is much faster than the gold standard gas chromatography (GC)-MS (typically > 30 min per sample). To demonstrate the power of this method, we performed site-saturation mutagenesis of the yeast fatty acid synthase and identified 9 mutants that resulted in improved MCFA production relative to the wild-type strain. This colony-based MALDI-ToF MS screening method offers an alternative way for engineering microbial fatty acid compositions in a high-throughput manner.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

All You Can Eat Yeast: Substituting Hexose Transporters With AtSWEET7 Alleviates Glucose Repression, Enabling Simultaneous Utilization of Sugars in Renewable Feedstocks

Yeast sugar transporters have highly evolved for preferential glucose transport, a significant roadblock for utilizing non-glucose sugars in renewable feedstocks such as lignocellulosic biomass. To enable simultaneous transport of multiple sugars, native hexose transporters were replaced by SWEET7p from Arabidopsis thaliana in engineered Saccharomyces cerevisiae capable of fermenting xylose. Engineered S. cerevisiae exhibited reduced glucose preference, simultaneously co-fermenting glucose, mannose, fructose, and xylose both in synthetic and industrial media. Continuous culture experiments demonstrated the co-consuming phenotype and alleviation of glucose repression by engineered S. cerevisiae. In addition to hexose and pentose, the NKSW7-1 strain consumed xylitol as a carbon source. Through transcriptomic and metabolomic analysis of the NKSW7-1 strain, we show that the replacement of HXT1-7 with AtSWEET7 led to systemwide reprogramming of the central carbon metabolism. This broad transport capacity of AtSWEET7p holds promise for achieving co-consumption of all sugars in underutilized renewable feedstocks by microbial cell factory.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Strategies to achieve high productivity, high conversion, and high yield in yeast fermentation of algal biomass hydrolysate

The conversion of carbohydrates in biomass via fermentation is an important component of an overall strategy to decarbonize the production of fuels and chemicals. Owing to the cost and resources required to produce biomass hydrolysates, the economic and environmental sustainability of these fermentation processes requires that they operate with high yields, sugar conversion, and productivity. Immobilized-cell technology in a continuous bioprocess can achieve significantly higher volumetric productivities than is possible from standard batch fermentation using free cells. Here, we demonstrate approaches for improvement of ethanol yield from algal hydrolysates and a mock hydrolysate medium. Saccharomyces cerevisiae was immobilized in alginate and incorporated into a two-column immobilized cell reactor system. Furthermore, the yeast quorum-sensing molecule, 2-phenylethanol, was added to improve ethanol yield by restricting growth and diverting sugar to ethanol. The bioreactor system could achieve high ethanol volumetric productivity (>20 g/ Lreactor ·h) and high glucose conversion (>99%) in mock hydrolysate, while the addition of 0.2% 2-phenylethanol resulted in 4.9% higher ethanol yield. With an algal hydrolysate of <10 g/L sugar, the ethanol volumetric productivity reached 9.8 g/ Lreactor ·h, and the addition of 0.2% 2-phenylethanol increased the ethanol yield by up to 7.4%. These results demonstrate the feasibility of novel strategies to achieve sustainability goals in biomass conversions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Fermented Cerasus humilis fruits protect against high‐fat diet induced hyperlipidemia which is associated with alteration of gut microbiota

Abstract BACKGROUND Hyperlipidemia is regarded as a public health matter, and its effective prevention and treatment are urgently required. However, the treatment of hyperlipidemia is still relatively scarce. RESULTS Fermented Cerasus humilis fruit (FCHF) had higher total flavonoid, total phenolic, procyanidin, and organic and free amino acid content, and lower total sugar content, than non‐fermented C. humilis fruit (NFCHF). Both FCHF and NFCHF treatment significantly prevent putting on weight. Furthermore, FCHF administration ameliorated hyperlipidemia and cholesterol over‐accumulation. In addition, FCHF administration activated the antioxidase system and decreased the malondialdehyde content to relieve oxidative stress, and showed more efficaciously than NFCHF administration. FCHF treatments significantly reverse the fat deposition in high‐fat diet rat liver. FCHF supplementation can relieve the dysbacteriosis induced by hyperlipidemia, and regulate the composition of rat gut microbiota by increasing the abundance of Prevotella and norank_f_Muribaculaceae . CONCLUSION Lactobacillus plantarum and Saccharomyces cerevisiae fermentation enhanced the antihyperlipidemic property of C. humilis fruits by promoting gut microbiota regulation. © 2022 Society of Chemical Industry.

Wang, Yu↗
DOE OSTI2023

The yeast ALA synthase C‐terminus positively controls enzyme structure and function

Abstract 5‐Aminolevulinic acid synthase (ALAS) is a pyridoxal 5′‐phosphate (PLP)‐dependent enzyme that catalyzes the first and rate‐limiting step of heme biosynthesis in α‐proteobacteria and several non‐plant eukaryotes. All ALAS homologs contain a highly conserved catalytic core, but eukaryotes also have a unique C‐terminal extension that plays a role in enzyme regulation. Several mutations in this region are implicated in multiple blood disorders in humans. In Saccharomyces cerevisiae ALAS (Hem1), the C‐terminal extension wraps around the homodimer core to contact conserved ALAS motifs proximal to the opposite active site. To determine the importance of these Hem1 C‐terminal interactions, we determined the crystal structure of S. cerevisiae Hem1 lacking the terminal 14 amino acids (Hem1 ΔCT). With truncation of the C‐terminal extension, we show structurally and biochemically that multiple catalytic motifs become flexible, including an antiparallel β‐sheet important to Fold‐Type I PLP‐dependent enzymes. The changes in protein conformation result in an altered cofactor microenvironment, decreased enzyme activity and catalytic efficiency, and ablation of subunit cooperativity. These findings suggest that the eukaryotic ALAS C‐terminus has a homolog‐specific role in mediating heme biosynthesis, indicating a mechanism for autoregulation that can be exploited to allosterically modulate heme biosynthesis in different organisms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Identification and expression of small multidrug resistance transporters in early‐branching anaerobic fungi

Abstract Membrane‐embedded transporters impart essential functions to cells as they mediate sensing and the uptake and extrusion of nutrients, waste products, and effector molecules. Promiscuous multidrug exporters are implicated in resistance to drugs and antibiotics and are highly relevant for microbial engineers who seek to enhance the tolerance of cell factory strains to hydrophobic bioproducts. Here, we report on the identification of small multidrug resistance (SMR) transporters in early‐branching anaerobic fungi (Neocallimastigomycetes). The SMR class of transporters is commonly found in bacteria but has not previously been reported in eukaryotes. In this study, we show that SMR transporters from anaerobic fungi can be produced heterologously in the model yeast Saccharomyces cerevisiae , demonstrating the potential of these proteins as targets for further characterization. The discovery of these novel anaerobic fungal SMR transporters offers a promising path forward to enhance bioproduction from engineered microbial strains.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Risk Assessment of Industrial Microbes Using a Terrestrial Mesocosm Platform

Abstract Industrial microbes and bio-derived products have emerged as an integral component of the bioeconomy, with an array of agricultural, bioenergy, and biomedical applications. However, the rapid development of microbial biotechnology raises concerns related to environmental escape of laboratory microbes, detection and tracking thereof, and resultant impact upon native ecosystems. Indeed, though wild-type and genetically modified microbes are actively deployed in industrial bioprocesses, an understanding of microbial interactivity and impact upon the environment is severely lacking. In particular, the persistence and sustained ecosystem impact of industrial microbes following laboratory release or unintentional laboratory escape remains largely unexplored. Herein, we investigate the applicability of soil-sorghum mesocosms for the ecological risk assessment of the industrial microbe, Saccharomyces cerevisiae . We developed and applied a suite of diagnostic and bioinformatic analyses, including digital droplet PCR, microscopy, and phylogenomic analyses to assess the impacts of a terrestrial ecosystem perturbation event over a 30-day time course. The platform enables reproducible, high-sensitivity tracking of S. cerevisiae in a complex soil microbiome and analysis of the impact upon abiotic soil characteristics and soil microbiome population dynamics and diversity. The resultant data indicate that even though S. cerevisiae is relatively short-lived in the soil, a single perturbation event can have sustained impact upon mesocosm soil composition and underlying microbial populations in our system, underscoring the necessity for more comprehensive risk assessment and development of mitigation and biocontainment strategies in industrial bioprocesses.

09 BIOMASS FUELS↗
DOE OSTI2022

Zng1 is a GTP-dependent zinc transferase needed for activation of methionine aminopeptidase

The evolution of zinc (Zn) as a protein cofactor altered the functional landscape of biology, but dependency on Zn also created an Achilles' heel, necessitating adaptive mechanisms to ensure Zn availability to proteins. A debated strategy is whether metallochaperones exist to prioritize essential Zn-dependent proteins. Here, we present evidence for a conserved family of putative metal transferases in human and fungi, which interact with Zn-dependent methionine aminopeptidase type I (MetAP1/Map1p/Fma1). Deletion of the putative metal transferase in Saccharomyces cerevisiae (ZNG1; formerly YNR029c) leads to defective Map1p function and a Zn-deficiency growth defect. In vitro, Zng1p can transfer Zn 2+ or Co 2+ to apo-Map1p, but unlike characterized copper chaperones, transfer is dependent on GTP hydrolysis. Proteomics reveal mis-regulation of the Zap1p transcription factor regulon due to loss of ZNG1 and Map1p activity, suggesting that Zng1p is required to avoid a compounding effect of Map1p dysfunction on survival during Zn limitation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Systematic identification of transcriptional activation domains from non-transcription factor proteins in plants and yeast

Transcription factors can promote gene expression through activation domains. Whole-genome screens have systematically mapped activation domains in transcription factors but not in non-transcription factor proteins (e.g., chromatin regulators and coactivators). To fill this knowledge gap, we employed the activation domain predictor PADDLE to analyze the proteomes of Arabidopsis thaliana and Saccharomyces cerevisiae. We screened 18,000 predicted activation domains from >800 non-transcription factor genes in both species, confirming that 89% of candidate proteins contain active fragments. Our work enables the annotation of hundreds of nuclear proteins as putative coactivators, many of which have never been ascribed any function in plants. Analysis of peptide sequence compositions reveals how the distribution of key amino acids dictates activity. Finally, we validated short, "universal" activation domains with comparable performance to state-of-the-art activation domains used for genome engineering. Our approach enables the genome-wide discovery and annotation of activation domains that can function across diverse eukaryotes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Discovery of additional ancient genome duplications in yeasts

Whole-genome duplication (WGD) has had profound macroevolutionary impacts on diverse lineages, preceding adaptive radiations in vertebrates, teleost fish, and angiosperms. In contrast to the many known ancient WGDs in animals, and especially plants, we are aware of evidence for only four WGDs in fungi. The oldest of these occurred ∼100 million years ago (mya) and is shared by ∼60 extant Saccharomycetales species, including the baker’s yeast Saccharomyces cerevisiae. Notably, this is the only known ancient WGD event in the yeast subphylum Saccharomycotina. The dearth of ancient WGD events in fungi remains a mystery. Some studies have suggested that fungal lineages that experience chromosome and genome duplication quickly go extinct, leaving no trace in the genomic record, while others contend that the lack of known WGDs is due to an absence of data. Under the second hypothesis, additional sampling and deeper sequencing of fungal genomes should lead to the discovery of more WGD events. Coupling hundreds of recently published genomes from nearly every described Saccharomycotina species, with three additional long-read assemblies, we discovered three novel WGD events. Although the functions of retained duplicate genes originating from these events are broad, they bear similarities to the well-known WGD that occurred in the Saccharomycetales. In conclusion, our results suggest that WGD may be a more common evolutionary force in fungi than previously believed.

convergent evolution↗
DOE OSTI2022

Evolution of Rev7 interactions in eukaryotic TLS DNA polymerase Polζ

Translesion synthesis (TLS) DNA polymerase Polζ is crucial for the bypass replication over sites of DNA damage. The Rev7 subunit of Polζ is a HORMA (Hop1, Rev7, Mad2) protein that facilitates recruitment of Polζ to the replication fork via interactions with the catalytic subunit Rev3 and the translesion synthesis scaffold protein Rev1. Human Rev7 (hRev7) interacts with two Rev7-binding motifs (RBMs) of hRev3 by a mechanism conserved among HORMA proteins whereby the safety-belt loop of hRev7 closes on the top of the ligand. The two copies of hRev7 tethered by the two hRev3-RBMs form a symmetric head-to-head dimer through the canonical HORMA dimerization interface. Recent cryo-EM structures reveal that Saccharomyces cerevisiae Polζ (scPolζ) also includes two copies of scRev7 bound to distinct regions of scRev3. Surprisingly, the HORMA dimerization interface is not conserved in scRev7, with the two scRev7 protomers forming an asymmetric head-to-tail dimer with a much smaller interface than the hRev7 dimer. Here, we validated the two adjacent RBM motifs in scRev3, which bind scRev7 with affinities that differ by two orders of magnitude and confirmed the 2:1 stoichiometry of the scRev7:Rev3 complex in solution. However, our biophysical studies reveal that scRev7 does not form dimers in solution either on its own accord or when tethered by the two RBMs in scRev3. These findings imply that the scRev7 dimer observed in the cryo-EM structures is induced by scRev7 interactions with other Polζ subunits and that Rev7 homodimerization via the HORMA interface is a mechanism that emerged later in evolution.

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

Unlocking the magic in mycelium: Using synthetic biology to optimize filamentous fungi for biomanufacturing and sustainability

Filamentous fungi drive carbon and nutrient cycling across our global ecosystems, through its interactions with growing and decaying flora and their constituent microbiomes. The remarkable metabolic diversity, secretion ability, and fiber-like mycelial structure that have evolved in filamentous fungi have been increasingly exploited in commercial operations. The industrial potential of mycelial fermentation ranges from the discovery and bioproduction of enzymes and bioactive compounds, the decarbonization of food and material production, to environmental remediation and enhanced agricultural production. Despite its fundamental impact in ecology and biotechnology, molds and mushrooms have not, to-date, significantly intersected with synthetic biology in ways comparable to other industrial cell factories (e.g. Escherichia coli,Saccharomyces cerevisiae, and Komagataella phaffii). In this review, we summarize a suite of synthetic biology and computational tools for the mining, engineering and optimization of filamentous fungi as a bioproduction chassis. A combination of methods across genetic engineering, mutagenesis, experimental evolution, and computational modeling can be used to address strain development bottlenecks in established and emerging industries. These include slow mycelium growth rate, low production yields, non-optimal growth in alternative feedstocks, and difficulties in downstream purification. In the scope of biomanufacturing, we then detail previous efforts in improving key bottlenecks by targeting protein processing and secretion pathways, hyphae morphogenesis, and transcriptional control. Bringing synthetic biology practices into the hidden world of molds and mushrooms will serve to expand the limited panel of host organisms that allow for commercially-feasible and environmentally-sustainable bioproduction of enzymes, chemicals, therapeutics, foods, and materials of the future.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Metabolic modulation: Pneumocystis phosphoglucomutase is a target influencing host recognition

Herein, this manuscript explores the significance of the phosphoglucomutase (PGM) enzyme in Pneumocystis spp., focusing on its role in fungal surface mannoprotein formation. Through expression of the Pneumocystis murina Pmpgm2 in a Saccharomyces cerevisiae pgm2Δ strain, we demonstrate restoration of binding to the mannose receptor (MR) and macrophages to wildtype yeast levels in this complemented strain. Gas Chromatography-Mass Spectroscopy (GC-MS) confirmed reduced mannose content in the pgm2Δ yeast strain compared to the wild-type and complemented Pmpgm2 cDNA-expressing strains. This study underscores fungal PGM function in dolichol glucosyl phosphate biosynthesis, crucial for proper cell wall mannoprotein formation. Furthermore, highlighting the conservation of targetable cysteine residues across fungal pathogens, PGM inhibition maybe a potential therapeutic strategy against a broad spectrum of fungal infections.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

The evolution of the GAL actose utilization pathway in budding yeasts

The Leloir galactose utilization or GAL pathway of budding yeasts, including that of the baker’s yeast Saccharomyces cerevisiae and the opportunistic human pathogen Candida albicans, breaks down the sugar galactose for energy and biomass production. Here, the GAL pathway has long served as a model system for understanding how eukaryotic metabolic pathways, including their modes of regulation, evolve. More recently, the physical linkage of the structural genes GAL1, GAL7, and GAL10 in diverse budding yeast genomes has been used as a model for understanding the evolution of gene clustering. In this review, we summarize exciting recent work on three different aspects of this iconic pathway’s evolution: gene cluster organization, GAL gene regulation, and the population genetics of the GAL pathway.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Comparative modeling reveals the molecular determinants of aneuploidy fitness cost in a wild yeast model

Although implicated as deleterious in many organisms, aneuploidy can underlie rapid phenotypic evolution. However, aneuploidy will be maintained only if the benefit outweighs the cost, which remains incompletely understood. To quantify this cost and the molecular determinants behind it, we generated a panel of chromosome duplications in Saccharomyces cerevisiae and applied comparative modeling and molecular validation to understand aneuploidy toxicity. We show that 74%–94% of the variance in aneuploid strains’ growth rates is explained by the cumulative cost of genes on each chromosome, measured for single-gene duplications using a genomic library, along with the deleterious contribution of small nucleolar RNAs (snoRNAs) and beneficial effects of tRNAs. Machine learning to identify properties of detrimental gene duplicates provided no support for the balance hypothesis of aneuploidy toxicity and instead identified gene length as the best predictor of toxicity. Our results present a generalized framework for the cost of aneuploidy with implications for disease biology and evolution.

genic load↗
DOE OSTI2020

A genetic toolbox for metabolic engineering of Issatchenkia orientalis

The nonconventional yeast Issatchenkia orientalis can grow under highly acidic conditions and has been explored for production of various organic acids. Yet, its broader application is hampered by the lack of efficient genetic tools to enable sophisticated metabolic manipulations. We recently constructed an episomal plasmid based on the autonomously replicating sequence (ARS) from Saccharomyces cerevisiae (ScARS) in I. orientalis and developed a CRISPR/Cas9 system for multiplexed gene deletions. Here we report three additional genetic tools including: (1) identification of a 0.8 kb centromere-like (CEN-L) sequence from the I. orientalis genome by using bioinformatics and functional screening; (2) discovery and characterization of a set of constitutive promoters and terminators under different culture conditions by using RNA-Seq analysis and a fluorescent reporter; and (3) development of a rapid and efficient in vivo DNA assembly method in I. orientalis, which exhibited ~100% fidelity when assembling a 7 kb-plasmid from seven DNA fragments ranging from 0.7 kb to 1.7 kb. As evidence of concept, we used these genetic tools to rapidly construct a functional xylose utilization pathway in I. orientalis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Improving CRISPR/Cas9-mediated genome editing efficiency in Yarrowia lipolytica using direct tRNA-sgRNA fusions

We report Yarrowia lipolytica is an important oleaginous yeast currently used in the production of specialty chemicals and has a great potential for further applications in lipid biotechnology. Harnessing the full potential of Y. lipolytica is, however, limited by its inherent recalcitrance to genetic manipulation. In contrast to Saccharomyces cerevisiae, Y. lipolytica is poor in homology-mediated DNA repair and thus in homologous recombination, which limits site-specific gene editing in this yeast. Recently developed CRISPR/Cas9-based methods using tRNA-sgRNA fusions succeeded in editing some genomic loci in Y. lipolytica. Nonetheless, the majority of other tested loci either failed editing or editing was achieved but at very low efficiency using these methods. Using tools of secondary RNA structure prediction, we were able to improve the design of the tRNA-sgRNA fusions used for the expression of single guide RNA (sgRNA) in such methods. This resulted in high efficiency CRISPR/cas9 gene editing at chromosomal loci that failed gene editing or were edited at very low efficiencies with previous methods. In addition, we characterized the gene editing performance of our newly designed tRNA-sgRNA fusions for both chromosomal gene integration and deletion. As such, this study presents an efficient CRISPR/Cas9-mediated gene-editing tool for efficient genetic engineering of Yarrowia lipolytica.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Establishing a versatile toolkit of flux enhanced strains and cell extracts for pathway prototyping

Building and optimizing biosynthetic pathways in engineered cells holds promise to address societal needs in energy, materials, and medicine, but it is often time-consuming. Cell-free synthetic biology has emerged as a powerful tool to accelerate design-build-test-learn cycles for pathway engineering with increased tolerance to toxic compounds. However, most cell-free pathway prototyping to date has been performed in extracts from wildtype cells which often do not have sufficient flux towards the pathways of interest, which can be enhanced by engineering. Here, in this study, to address this gap, we create a set of engineered Escherichia coli and Saccharomyces cerevisiae strains rewired via CRISPR-dCas9 to achieve high-flux toward key metabolic precursors; namely, acetyl-CoA, shikimate, triose-phosphate, oxaloacetate, α-ketoglutarate, and glucose-6-phosphate. Cell-free extracts generated from these strains are used for targeted enzyme screening in vitro. As model systems, we assess in vivo and in vitro production of triacetic acid lactone from acetyl-CoA and muconic acid from the shikimate pathway. The need for these platforms is exemplified by the fact that muconic acid cannot be detected in wildtype extracts provided with the same biosynthetic enzymes. We also perform metabolomic comparison to understand biochemical differences between the cellular and cell-free muconic acid synthesis systems (E. coli and S. cerevisiae cells and cell extracts with and without metabolic rewiring). While any given pathway has different interfaces with metabolism, we anticipate that this set of pre-optimized, flux enhanced cell extracts will enable prototyping efforts for new biosynthetic pathways and the discovery of biochemical functions of enzymes.

59 BASIC BIOLOGICAL SCIENCES↗